Lung fibroblast-specific molecular guidance system peptides and uses thereof

Abstract

Embodiments are directed to Molecular Guidance System (MGS) peptides, MGS compounds comprising at least two MGS peptides, MGS-cargo conjugates comprising an MGS peptide conjugated to a cargo, directly or indirectly through a linker, and methods of using such MGS peptides, MGS compounds, and MGS-cargo conjugates. An example Molecular Guidance System (MGS) peptide is selected from SEQ ID NO: 1-29.

Description

PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)LUNG FIBROBLAST-SPECIFIC MOLECULAR GUIDANCE SYSTEM PEPTIDES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 598,316, filed on November 13, 2023, which is incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The contents of the electronic sequence listing(S164719111 l_SequenceListing.xml; Size: 70,727 bytes; and Date of Creation: November 12, 2024) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Lung fibroblasts constitute a small percentage of cells present in lung tissue.However, lung fibroblasts play an important role in the development and maintenance of the structure of the lung. For example, lung fibroblasts produce proteins that drive the formation of the connective tissue. These cells are also involved in the development of inflammatory diseases of the lung, such as pulmonary fibrosis and emphysema. In such diseases, normal lung tissue is replaced by connective tissue and loses the ability to support the exchange of gases between blood and air. Pulmonary fibrosis can be initiated by a number of mechanisms which involve inflammation and activation of fibroblast genes that produce scar-like connective tissue. Thus, it is of interest to target lung fibroblasts, without impacting other cell types.

[0004] Molecular Guidance System (MGS) peptides are a class of safe and efficacious intracellular targeting peptides which can be used to intracellularly deliver cargo. MGS combination therapeutics can create an entirely new market impacting virtually every disease state and creating a major breakthrough in modern medicine. Delivering cargo to lung fibroblasts can be used to halt progression of inflammation and activation of fibroblast genes that produce scar-like connective tissue.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)SUMMARY

[0005] The present invention relates to Molecular Guidance System (MGS) peptides, MGS compounds, and MGS-cargo conjugates for the selective delivery of cargo to lung fibroblasts.

[0006] Various aspects of the present disclosure are directed to a Molecular Guidance System (MGS) peptide selected from SEQ ID NOs: 1-29.

[0007] Various aspects are directed to a Molecular Guidance System (MGS) peptide selected from SEQ ID NOs: 1-2, 7-8, 10, 15, 19, 21, and 23-29.

[0008] In some aspects, the MGS peptide is selected from SEQ ID NOs: 1-2, 7-8, 10, 15, 19, 21, 24, and 27-28.

[0009] In some aspects, the MGS peptide selectively binds to lung fibroblasts.

[0010] In some aspects, the MGS peptide comprises a protecting group on the N- terminus.

[0011] In some aspects, the protecting group is selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0012] In some aspects, the protecting group is selected from: -COCH3, -COH, -CO- (CH)n-CHs, -CO-(CH)n-NH2, y-Aminobutyric acid, pyro-glutamic acid, methyl, -CO- (CH)2-CO-OH, -CO-(CH)2-CO-COH3, -CO-polyethylene glycol (PEG)X-(CH)2- NH2, - CO-PEGX-(CH)2-COH3, and CO-PEGX-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

[0013] In some aspects, the MGS peptide is selected from SEQ ID NOs: 30-58.

[0014] Various aspects of the present disclosure are directed to a Molecular Guidance System (MGS) compound comprising at least two MGS peptides, wherein the at least two MGS peptides are each independently selected from SEQ ID NOs: 1-29.

[0015] In some aspects, each of the at least two MGS peptides comprise a protecting group on the N-terminus selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0016] In some aspects, the at least two MGS peptides comprise two MGS peptides.

[0017] In some aspects, the at least two MGS peptides comprise four MGS peptides.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0018] In some aspects, the at least two MGS peptides are each the same or each MGS peptide of the at least two MGS peptides is different from one another.

[0019] In some aspects, the MGS compound further comprises a linker structure comprising linkers selected from a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

[0020] In some aspects, the linker structure comprises: at least two PEG linkers, each respectively conjugated to one of the at least two MGS peptides, directly or indirectly; and at least one branch linker conjugated to two of the at least two PEG linkers, directly or indirectly.

[0021] In some aspects, the branch linker comprises a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0022] In some aspects, the MGS compound further comprises a reactive group conjugated to the linker structure, directly or indirectly, and to allow for conjugation to a cargo.

[0023] In some aspects, the reactive group is selected from: a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, a maleimide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

[0024] In some aspects, the linker structure further comprises additional linkers comprising reactive groups and the at least two PEG linkers are indirectly conjugated to, respectively, the one of the at least two MGS peptides through the reactive groups.

[0025] Various aspects of the present disclosure are directed to a Molecular Guidance System (MGS)-cargo conjugate comprising an MGS peptide conjugated to a cargo,PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) directly or indirectly through a linker, wherein the MGS peptide is selected from SEQ ID NOs: 1-58.

[0026] In some aspects, the MGS peptide is selected from SEQ ID NOs: 1-29, and the MGS peptide further comprises a protecting group on the N-terminus selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0027] In some aspects, the MGS-cargo conjugate comprises a multimer that includes the MGS peptide and at least one additional MGS peptide, each of the MGS peptide and at least one additional MGS peptide being independently selected from SEQ ID NOs: 1- 58.

[0028] Various aspects of the present disclosure are directed to a Molecular Guidance System (MGS) peptide comprising SB1WB2B3B4NDIYD (SEQ ID NO: 59), wherein: Bi is selected from A and F; B2 is selected from A and M; B3 is selected from A and I; and B4 is selected from A and L.

[0029] In some aspects, the MGS peptide further comprises B5B6PB7B8B9B10B11B12 such that the MGS peptide comprises SB1WB2B3B4NDIYDB5B6PB7B8B9B10B11B12 (SEQ ID NO: 60), wherein: Bs is selected from A and D; Be is selected from A and T; B7 is selected from A and L; Bs is selected from A and S; B9 is selected from A and E; Bio is selected from A and F; Bn is selected from A and R; and B12 is selected from A and L.

[0030] In some aspects, the MGS peptide selectively binds to lung fibroblasts.

[0031] In some aspects, the MGS peptide comprises a protecting group on the N- terminus.

[0032] In some aspects, the protecting group is selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0033] In some aspects, the protecting group is selected from: -COCH3, -COH, -CO- (CH)n-CH3, -CO-(CH)n-NH2, y-Aminobutyric acid, pyro-glutamic acid, methyl, -CO- (CH)2-CO-OH, -CO-(CH)2-CO-COH3, -CO-uolyethylene glycol (PEG)X-(CH)2- NH2, - CO-PEGX-(CH)2-COH3, and CO-PEGX-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0034] Various aspects of the present disclosure are directed to a Molecular Guidance System (MGS) compound comprising at least two MGS peptides of any of claims 23- 28.

[0035] Various aspects of the present disclosure are directed to a Molecular Guidance System (MGS)-cargo conjugate comprising an MGS peptide of any of claims 23-28 conjugated to a cargo, directly or indirectly through a linker.

[0036] Various aspects of the present disclosure are directed to a method of targeting lung fibroblasts in a subject comprising administering the MGS compound or MGS- cargo conjugate of any one of claims 9-30 to a subject.

[0037] In some aspects, the MGS peptide preferentially binds to lung fibroblasts and internalizes the cargo to the lung fibroblasts.

[0038] In some aspects, the cargo retains functional activity inside the lung fibroblasts.

[0039] In some aspects, administering comprises an intravenous or intrathecal administration of a formulation comprising the MGS-cargo conjugate.BRIEF DESCRIPTION OF THE DRAWINGSVarious example embodiments can be more completely understood in consideration of the following detailed description in connection with the accompanying drawings, in which:

[0040] FIGs. 1 A-1E illustrate example Molecular Guidance System (MGS) compounds and MGS-cargo conjugates comprising an MGS peptide, in accordance with the present disclosure.

[0041] FIGs. 2A-2B show the analytical reverse-phase high-performance liquid chromatography (RP-HPLC) (FIG. 2A) and electrospray ionization Mass Spectrometry (MS) (FIG. 2B) results of the MGS NHLF V1-2 B structure.

[0042] FIGs. 3 A-3D show the quantitative flow cytometry assay results for MGS peptide internalization gating.

[0043] FIGs. 4A-4B show the quantitative flow cytometry assay results for MGS peptide internalization including the set of standardization beads.

[0044] FIG. 5 is a graph showing the concentration curve and ECso of MGS NHLF Vl- 2_B after one hour incubation with normal human lung fibroblasts (NHLF).PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0045] FIGs. 6A-6B are fluorescent images of control incubations and FIG. 6C shows cellular internalization of MGS peptides by NHLF.

[0046] FIGs. 7A-7B show the analytical RP-HPLC (FIG. 7A) and electrospray ionization MS (FIG. 7B) results of the MGS_NHLF_V1-4_AF647 structure.

[0047] FIG. 8 is a graph showing MGS NHLF V1-4 AF647 uptake by NHLF.

[0048] FIG. 9 is a graph showing the internalization of MGS_NHLF_V1-4_AF647 by NHLF.

[0049] FIGs. 10 A- 10C show the flow cytometry results indicating internalized MGS_NHLF_V1-4_AF647 peptides escape from vesicles to cytoplasm.

[0050] FIGs. 11 A-l IB are microscopy images of NHLF samples after incubation with AF647 control (FIG. 11 A) and MGS NHLF V1-4 AF647 (FIG. 1 IB).

[0051] FIGs. 12A-12D are four versions of the same microscopy image and associated histograms showing the MGS_NHLF_V1-4_AF647 cells in vesicles to highlight the intensity distribution of pixels where AF647 is detected in the cell.

[0052] FIGs. 13A-13C are microscopy images of NHLF alone (FIG. 13A), NHLF incubated with AF647 (FIG. 13B), and NHLF incubated with MGS NHLF Vl- 4 AF647 (FIG. 13C), which were imaged after one hour incubation.

[0053] FIGs. 14A-14C are the same microscopy images from FIGs. 13A-13C of NHLF alone (FIG. 14 A), NHLF incubated with AF647 (FIG. 14B), and NHLF incubated with MGS NHLF V1-4 AF647 (FIG. 14C) but with a change in setting of the intensity threshold to enable visualization of modest intensity pixels (e.g., above background) that are outside of intracellular vesicles.

[0054] FIGs. 15A-15D are microscopy images of NHLF after incubation with MGS NHLF V1-4 AF647 and which are adjusted to show pixels above different thresholds.

[0055] FIG. 16 is a graph showing internalization of MGS_NHLF_V1-4_AF647 by LL29 cells.

[0056] FIG. 17 is a graph showing internalization of MGS_NHLF_V1-4_AF647 by mouse pulmonary fibroblasts (MPF) cells.

[0057] FIGs. 18A-18F are graphs showing various different MGS peptide uptake by NHLF.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0058] FIG. 19 shows the analytical RP-HPLC (left and middle) and electrospray ionization MS (right) results of the MGS_NHLF_V1-4_AF647 structure after incubation in 50% human serum for various times.

[0059] FIGs. 20A-20D show details of human serum stability assessment results of MGS_NHLF_V29-4_AF647.

[0060] FIGs. 21 A-21D show details of mouse serum stability assessment results of MGS NHLF V2-4 AF647.

[0061] FIG. 22A-22C show details of mouse serum stability assessment results of MGS NHLF V29-4 AF647.

[0062] FIGs. 23A-23E show example fragments of MGS NHLF V2-4 AF647 that arose during incubation of MGS_NHLF_V2-4_AF647 with mouse serum.

[0063] FIGs. 24A-24E show example fragments of MGS NHLF V29-4 AF647 that arose during incubation of MGS_NHLF_V29-4_AF647 with mouse serum.

[0064] FIG. 25 shows images of heart, lung, and spleen from an untreated mouse (control) and mice treated with MGS_NHLF_V2-4_AF647.

[0065] FIG. 26 shows images of heart, lung, and spleen from an untreated mouse (control) and mice treated with MGS_NHLF_V29-4_AF647.

[0066] FIG. 27 is a summary graph showing the ex vivo MGS peptide distribution in isolated organs as imaged by IVIS near infrared (NIR).

[0067] FIGs. 28A-28F show the results of flow cytometry gating of single cells which were isolated from mouse lung tissue.

[0068] FIGs. 29A-29B show the results of flow cytometry from lung fibroblasts (PEneg / FITCp°s cells) isolated from mouse lung tissue collected twenty-four hours after injection with MGS_NHLF_V2-4_AF647 (FIG. 29 A) and MGS_NHLF_V29-4_AF647 (FIG. 29B).

[0069] FIGs. 30A-30B show the results of flow cytometry from unknown cells (PEneg / FITCneg) isolated from mouse lung tissue collected twenty-four hours after injection with MGS_NHLF_V2-4_AF647 (FIG. 30 A) and MGS_NHLF_V29-4_AF647 (FIG. 30B).

[0070] FIGs. 31A-31C show an example structure of MGS_NHLF_V8-4_AF647 and analytical results of the structure.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191.l l l (SRI-230091)

[0071] FIGs. 32A-32C show an example structure of MGS NHLF V9-4 AF647 and analytical results of the structure.

[0072] FIG. 33 shows an example structure of MGS NHLF V10-4 AF647.

[0073] FIGs. 34A-34C show an example structure of MGS NHLF Vl 1-4 AF647 and analytical results of the structure.

[0074] FIG. 35 shows an example structure of MGS NHLF V12-4 AF647.

[0075] FIG. 36 shows an example structure of MGS NHLF V13-4 AF647.

[0076] FIGs. 37A-37C show an example structure of MGS NHLF V14-4 AF647 and analytical results of the structure.

[0077] FIGs. 38A-38C show an example structure of MGS NHLF V15-4 AF647 and analytical results of the structure.

[0078] FIGs. 39A-39C show an example structure of MGS NHLF V16-4 AF647 and analytical results of the structure.

[0079] FIG. 40 shows an example structure of MGS NHLF V17-4 AF647.

[0080] FIGs. 41A-41C show an example structure of MGS NHLF V18-4 AF647 and analytical results of the structure.

[0081] FIGs. 42A-42C show an example structure of MGS NHLF V19-4 AF647 and analytical results of the structure.

[0082] FIGs. 43A-43C show an example structure of MGS NHLF V20-4 AF647 and analytical results of the structure.

[0083] FIG. 44 shows an example structure of MGS NHLF V21-4 AF647.

[0084] FIGs. 45A-45C show an example structure of MGS NHLF V22-4 AF647 and analytical results of the structure.

[0085] FIG. 46 shows an example structure of MGS NHLF V23-4 AF647.

[0086] FIG. 47 shows an example structure of MGS NHLF V28-4 AF647.

[0087] FIGs. 48A-48C show an example structure of MGS NHLF V29-4 AF647 and analytical results of the structure.DETAILED DESCRIPTION

[0088] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specificPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) examples in which the disclosure can be practiced. It is to be understood that other examples can be utilized, and various changes may be made without departing from the scope of the disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the disclosure is defined by the appended claims. It is to be understood that features of the various examples described herein may be combined, in part or whole, with each other, unless specifically noted otherwise.

[0089] As described above, lung fibroblasts are a type of cell present in lung tissue and can drive the formation of connective tissue. These cells are involved in the development of inflammatory lung diseases, including pulmonary fibrosis and emphysema. Embodiments of the present disclosure are directed to Molecular Guide System (MGS) peptides and compounds containing MGS peptides that specifically bind to and are internalized by lung fibroblasts, but not other types of cells. The MGS peptides can be conjugated to cargo to form an MGS-cargo conjugate, where the cargo can turn off the expression of the connective tissue proteins by the lung fibroblasts and / or halt progression of the disease, among other applications. Various embodiments are directed to MGS peptides that target normal human lung fibroblasts and the optimization of MGS peptides, which can be used to target lung fibroblasts in vivo.

[0090] Pulmonary fibrosis is a lethal disease, with patients generally living for three to five years from diagnosis. Current treatments, such as oxygen therapy, removal of sources of inflammation, inhibitors that slow down scar formation, and lung transplantation, are supportive. There are no current reagents that specifically target lung fibroblasts. Embodiments of the present disclosure, which include MGS peptides that bind to and are internalized by the lung fibroblasts, can enable novel treatments for pulmonary fibrosis and other diseases associated with lung fibroblasts, such as via delivery of therapeutics directly to the lung fibroblasts to block the expression of proteins that drive fibrosis. In some embodiments, cargo comprising therapeutic molecules can be conjugated (e.g., chemically linked) to the MGS peptides and delivered to the lung fibroblasts directly. This can allow for specific and potent therapies with fewer side effects. For example, the therapeutic cargo can be delivered by the MGS peptide and can block the expression of fibrosis-promoting proteins by the lung fibroblasts.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0091] The foregoing peptides, compounds, conjugates, and method embodiments have been provided in sufficient detail, but it is not the intention of the application for the disclosed embodiments to be limiting. Additional adaptations and / or modifications are possible, and, in broader aspects, these adaptations and / or modification are also encompassed. Accordingly, departure may be made from the foregoing embodiments without departing from the spirit of the peptides, compounds, conjugates, and / or methods.

[0092] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a composition is disclosed and discussed and a number of modifications that can be made to a number of molecules including the MGS peptide are discussed, each and every combination and permutation of the compositions and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment orPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0093] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0094] It must be noted that as used herein and in the appended claims, the singular forms "a ", "an", and "the", as well as “one or more” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "an MGS peptide" includes a plurality of such MGS peptides, such as two, three, four, or more, reference to "the cargo " is a reference to one or more cargo and equivalents thereof known to those skilled in the art, and so forth.

[0095] As used herein, “treat” is meant to mean administer a composition of the invention to a subject, such as a human or other mammal (for example, an animal model), that has a disease or condition, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease or condition. In some embodiments, the disease or condition is fibrosis and emphysema. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment comprises delivery of one or more of the disclosed compositions to a subject.

[0096] As used herein, “prevent” is meant to mean minimize the chance that a subject who has an increased susceptibility for developing disease, disorder or condition will develop the disease, disorder, or condition.

[0097] As used herein, the term "subject" refers to or includes the target of administration, e.g., a human. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat,PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) guinea pig, fruit fly, etc.). In some embodiments, a subject is a mammal. In some embodiments, a subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent, and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

[0098] As used herein, the term "patient" refers to or includes a subject afflicted with a disease or disorder. For example, the term "patient" includes human and veterinary subjects. In some embodiments, the “patient” has been diagnosed with a need for treatment prior to the administering step.

[0099] As used herein, the term "amino acid sequence" refers to or includes a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; and Nle, norleucine. Amino acids can include modified forms of W, G, and A. The modified forms of W, G, and A can include, but are not limited to, a-t-butylglycine, 2-Aminoisobutyric acid (Aib), and modified tryptophan, including -CN, -OH, -Cl, -F, -CH3, or -OCH3 modifications in position 5, 6, or 7 of the indole ring, among other modifications on the indole ring.

[0100] “Polypeptide" as used herein refers to or includes any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

[0101] In addition, as used herein, the term "peptide" refers to or includes amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and can contain modified amino acids other than the 20 standard protein building block amino acids. The peptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sitesPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) in a given peptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins — Structure and Molecular Properties 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)). N-terminus of a peptide (or amino acid or other molecule) refers to or includes an end with a free amino group. The C-terminus of a peptide (or amino acid or other molecule) refers to or includes an end with a free carboxyl group.

[0102] As used herein, “MGS peptide” refers to or includes a peptide which binds to target cells and is capable of being conjugated to cargo and mediating internalization of the cargo to the target cell. “Cargo” refers to or includes molecules, compounds, or other structures which are capable of being conjugated to the MGS peptide and being delivered to the target cells. Cargo can be interchangeably referred to as “a cargo moiety”, “a cargo molecule”, or “a cargo compound”. Example cargo can include, without limitation, a nucleic acid, a peptide, a protein, an antibody, a lipid, an imaging agent, a dye, a therapeutic, a small molecule, a radionuclide, a carbohydrate, a nanoparticle, or any combination thereof.

[0103] The phrase “nucleic acid” or “nucleic acid sequence”, as used herein, refers to or includes a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester intemucleoside linkages (e.g., peptidePCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, siRNA, mRNA, miRNA, or any combination thereof.

[0104] As used herein, a “reactive group” refers to or includes an atom, group of atoms, molecule, compound, or other structure that is capable of undergoing a chemical reaction with another group (e.g., moiety). In some embodiments, a reactive group can be or form part of a linker. In some embodiments, a reactive group can facilitate conjugation of a particular cargo and can be chosen for such facilitation. The reactive group(s) can be used to link MGS peptides to other linkers, linkers to linkers, and / or linkers to cargo. A “linker” refers to or includes a molecule, compound, or other structure which can be used to link (e.g., by covalent bond) at least two components of a composition. As used herein, the reactive group can be interchangeably referred to as “a reactive moiety”, “a reactive molecule”, or “a reactive compound”. The linker can be interchangeably referred to as “a linker moiety” or “a linker molecule”.

[0105] As used herein, “MGS compound” refers to or includes a compound comprising an MGS peptide and at least one additional molecule. Example MGS compounds include an MGS peptide and a linker or linker structure, at least two MGS peptides, and / or at least two MGS peptides and a linker or linker structure, among other examples. “MGS-cargo conjugate” refers to or includes a compound comprising least one MGS peptide and cargo which are joined together (e.g., conjugated) either directly or indirectly through a linker. Unless expressly stated otherwise, when used herein, “conjugated to” refers to both direct and indirect conjugations, whether or not “directly or indirectly” is expressly recited.

[0106] As used herein, “effective amount” of a compound or conjugate is meant to mean a sufficient amount of the compound or conjugate to provide the desired effect. The exact amount required can vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of disease (or underlying genetic defect) that is being treated, the particular compound or conjugate used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0107] As used herein, “selectively binds”, “specifically binds”, and “preferentially binds” is meant that a nucleic acid (e.g., cargo) or MGS peptide recognizes and physically interacts with its target (for example, a specific cell type) and does not significantly recognize and interact with other targets.

[0108] The term "percent (%) homology" is used interchangeably herein with the term "percent (%) identity" and refers to or includes the level of nucleic acid or amino acid sequence identity when aligned with a wild type sequence or sequence of interest using a sequence alignment program. For example, as used herein, 80% homology means the same thing as 80% sequence identity determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence identity over the length of the given sequence. Example levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity to a given sequence, e.g., any of the MGS peptide sequences, as described herein. Example computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet. See also, Altschul, et al., 1990 and Altschul, et al., 1997. Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program can be used for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (See, e.g., Altschul, S. F., et al., Nucleic Acids Res.25:3389-3402, 1997). A preferred alignment of selected sequences in order to determine "% identity" between two or more sequences, is performed using for example, the CLUSTAL-W program in Mac Vector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0109] Substitutions, deletions, insertions, or any combination thereof can be used to arrive at a final derivative, variant, or analog. Generally, these changes are performed onPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) a few nucleotides to minimize the alteration of the molecule. However, larger changes can be tolerated in certain circumstances.

[0110] Generally, the nucleotide identity between individual variant sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a “variant sequence” can be one with the specified identity to the parent or reference sequence (e.g., wild-type sequence) of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent sequence. For example, a “variant sequence” can be a sequence that contains 1, 2, or 3, 4 nucleotide base changes as compared to the parent or reference sequence of the invention, and shares or improves biological function, specificity and / or activity of the parent sequence. Thus, a “variant sequence” can be one with the specified identity to the parent sequence of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent sequence. The variant sequence can also share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of a reference sequence (e.g., an MGS peptide sequence).

[0111] As used herein, "modulate" is meant to mean to alter, by increasing or decreasing.

[0112] “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0113] Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specificallyPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0114] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0115] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0116] Disclosed herein are MGS peptides, MGS compounds, and MGS-cargo conjugates comprising MGS peptide(s) that selectively and / or specifically bind to lung fibroblasts. For example, the MGS peptides of the present disclosure selectively bind to lung fibroblasts, such as human lung fibroblasts. Example MGS peptides include, but are not limited to, the MGS sequences in Table 1. Other examples are directed to methods comprising at least one MGS peptide and methods of using example MGS peptides, MGS compounds, and / or MGS-cargo conjugates.

[0117] In some embodiments, an MGS peptide comprises SB1WB2B3B4NDIYD (SEQ ID NO: 59). In some embodiments, Bi is selected from A and F. In some embodiments, B2 is selected from A and M. In some embodiments, B3 is selected from A and I. And, in some embodiments, B4 is selected from A and L.

[0118] In some embodiments, the MGS peptide further includes B5B6PB7B8B9B10B11B 12 such that the MGS peptide comprises SB1WB2B3B4NDIYDB5B6PB7B8B9B10B11B12 (SEQ ID NO: 60). In some embodiments, Bs is selected from A and D. In some embodiments, Be is selected from A and T. In some embodiments, B7 is selected from A and L. In some embodiments, Bs is selected from A and S. In some embodiments, B9 is selected from A and E. In some embodiments, Bio is selected from A and F. In some embodiments, Bn is selected from A and R. In some embodiments, B12 is selected from A and L. In some embodiments, at least one of amino acids in the MGS peptide can include a d-amino acid form of the listed amino acid, as represented by “d(X)”.

[0119] In some embodiments, a MGS peptide is selected from SFWMILNDIYDDTPLSEFRL (SEQ ID NO: 1), SFWMILNDIYDDTPLSAAAA (SEQ ID NO: 2), SFWMILNDIYDDAAAAEFRL (SEQ ID NO: 3), SFWMILNDAAAATPLSEFRL (SEQ ID NO: 4), SFWMAAAAIYDDTPLSEFRL (SEQ ID NO: 5), AAAAILNDIYDDTPLSEFRL (SEQ ID NO: 6), SFWMILNDIYDDTPLAEFRL (SEQ ID NO: 7), SFWMILNDIYDDTPASEFRL (SEQ ID NO: 8), SFWMILNDIYDDTALSEFRL (SEQ ID NO: 9), SFWMILNDIYDDAPLSEFRL (SEQ ID NO: 10), SFWMILNAIYDDTPLSEFRL (SEQ ID NO: 11), SFWMILADIYDDTPLSEFRL (SEQ ID NO: 12), SFWMIANDIYDDTPLSEFRL (SEQ ID NO: 13), SFWMALNDIYDDTPLSEFRLPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)(SEQ ID NO: 14), SFWMILNDIYDATPLSEFRL (SEQ ID NO: 15), SFWMILNDIYADTPLSEFRL (SEQ ID NO: 16), SFWMILNDIADDTPLSEFRL (SEQ ID NO: 17), SFWMILNDAYDDTPLSEFRL (SEQ ID NO: 18), SFWAILNDIYDDTPLSEFRL (SEQ ID NO: 19), SFAMILNDIYDDTPLSEFRL (SEQ ID NO: 20), SAWMILNDIYDDTPLSEFRL (SEQ ID NO: 21), AFWMILNDIYDDTPLSEFRL (SEQ ID NO: 22), SFWMILNDIYDDTPLSEFRA (SEQ ID NO: 23), SFWMILNDIYDDTPLSEFAL (SEQ ID NO: 24), SFWMILNDIYDDTPLSEARL (SEQ ID NO: 25), SFWMILNDIYDDTPLSAFRL (SEQ ID NO: 26), SFWAIANDIYD (SEQ ID NO: 27), SFWAIANDIYDDTPLSEFRL (SEQ ID NO: 28), and SFWAIANDIYDDTPLSEFd(R)L (SEQ ID NO: 29).

[0120] As further described below, any of the described MGS peptides can be modified. For example, the MGS peptide can be modified at the N-terminus by acetylation or other alkylation, cyclization, amino-alkylation, methylation, succinylation, pegylation, and combinations thereof. In any such embodiment, the MGS peptide can be referred to as being acetylated, alkylated, cyclated, amino-alkylated, methylated, succinylated, and / or pegylated. In such embodiments, the MGS peptide comprises a protecting group on the N-terminus. In some embodiments, the N-terminal protecting group can comprise an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof. In some embodiments, the MGS peptide comprises any of SEQ ID NOs: 30-58.

[0121] In some embodiments, the protecting group on the N-terminus of the MGS peptide can comprise -COCH3, -COH, -CO-(CH)n-CH3, -CO-(CH)n-NH2, y- Aminobutyric acid, pyro-glutamic acid, methyl, -CO-(CH)2-CO-OH, -CO-(CH)2-CO- COH3, -CO-PEGX-(CH)2- NH2, -CO-PEGX-(CH)2-COH3, or CO-PEGx-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36. For example, the protecting group can be selected from:-20,PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)4. In the above-listing and non-limiting example protecting groups, the -NH group (on right) represents the free amino group of the MGS peptide and the -NH does not form part of the protecting group.

[0122] In some embodiments, the MGS peptide can comprise any of SEQ ID NOs: 1- 60 as shown below in Table 1. In some embodiments, the MGS peptide can comprise any of SEQ ID NOs: 1-58, SEQ ID NOs: 1-30, SEQ ID NOs: 1-29, SEQ ID NOs: 1-2, 7-8, 10, 15, 19, 21, and 23-29, or SEQ ID NOs: 1-2, 7-8, 10, 15, 19, 21, 24, and 27-28, among other combinations.Table 1 : Example MGS peptide sequencesPCT / US24 / 5564213 November 2024 (13.11.2024)S1647.191.lll (SRI-230091)PCT / US24 / 5564213 November 2024 (13.11.2024)S1647.191.lll (SRI-230091)PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0123] In some embodiments, the MGS peptide can selectively bind to lung fibroblasts. For example, MGS peptides of the present disclosure can have high affinity and specificity for lung fibroblasts to allow for delivery of cargo into the lung fibroblasts. In some embodiments, the MGS peptides comprising any of SEQ ID NOs: 1-60 showed affinity to a lung fibroblast and internalized into the lung fibroblast. In some embodiments, the lung fibroblasts can include human lung fibroblasts, such as normal human lung fibroblasts. Normal human lung fibroblasts, as used herein, refer to or include lung fibroblasts that exhibit normal growth and typical cell life cycles, such as those that do not otherwise exhibit features of disease or other conditions.Embodiments are not so limited and the lung fibroblasts can be from other animals.

[0124] Various embodiments are directed to MGS-cargo conjugates and MGS compounds comprising an MGS peptide. In some embodiments, the MGS cargoconjugates and / or MGS compounds comprising the MGS peptide further comprise aPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) linker, such as further illustrated by the conjugates and compounds illustrated by FIGs. 1 A-1E. Example linkers include a PEG linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, and a reactive group, as well as combinations thereof. In some embodiments, the linker is conjugated to the C-terminus of the MGS peptide. Accordingly, in some embodiments, the MGS-cargo conjugate and / or MGS compound comprising the MGS peptide can comprise any of the sequences set forth in SEQ ID NOs: 1-60, wherein SEQ ID NOs: 1-60 can be conjugated to PEG and / or another linker on the C-terminus of the MGS peptide.

[0125] In some embodiments, the linker can be any of those as further described herein. For example, a linker can be any length that allows conjugation of an MGS peptide with something else and prevents steric hindrance.

[0126] In some embodiments, the linker comprises a PEG linker. A PEG linker, as used herein, refers to or includes a linker containing at least one PEG unit. In some embodiments, the PEG linker can comprise a plurality of PEG units. For example, the number of PEG units in the PEG linker can be between 1-24 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the PEG linker comprises PEGn or PEG12. In any such embodiment, the MGS peptide can be referred to as being pegylated.

[0127] In some embodiments, the MGS peptide can be covalently attached to a cargo, directly or indirectly through a linker. Such cargo can include nucleic acids, small molecules, antibodies, proteins, lipids, imaging agents, dyes, radionuclides, carbohydrates, nanoparticles, and other cargo which can be used for therapeutic purposes. For example, in embodiments including a PEG linker, the number of PEG units can be of sufficient length to separate the MGS peptide from the cargo to prevent any steric interference between the MGS peptide and the cargo.

[0128] In some embodiments, the versatile chemistry of the MGS peptide sequence allows for modification(s) that enhances the lung fibroblast-targeting sensitivity and specificity, as well as the combination with cargo. In some embodiments, the MGS peptide has the capability to induce lung fibroblast targeting sensitivity and specificity. Thus, the MGS peptide can be used as a guiding system for targeting lung fibroblasts.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0129] Table 1 illustrates monomer MGS peptides. In some embodiments, the MGS peptides shown in Table 1 can be used to form multimers, such as dimers, trimers, and tetramers. As sometimes used herein, adding a “-2” at the end of the MGS peptide name refers to a dimer and adding a “-4” at the end of the MGS peptide name refers to a tetramer. For example, MGS NHLF V2-2 refers to the dimeric version of the CH3CO - SFWMILNDIYDDTPLSEFRL (SEQ ID NO: 30) sequence. As another example, MGS_ NHLF V2-4 refers to the tetrameric version of the CH3CO - SFWMILNDIYDDTPLSEFRL (SEQ ID NO: 30) sequence.

[0130] Embodiments are not limited to multimers which comprise copies of the same MGS peptides. In some embodiments, a multimer can include a copy or copies of at least two MGS peptides. For example, a dimer can include a first MGS peptide and a second MGS peptide that is different than the first MGS peptide. As another example, a tetramer can include a first MGS peptide, a second MGS peptide, a third MGS peptide, and a fourth MGS peptide, wherein each of the first, second, third, and fourth MGS peptides are different from one another. As a further example, a tetramer can include two copies of a first MGS peptide and two copies of a second MGS peptide that is different from the first MGS peptide. As a further example, a tetramer can include three copies of a first MGS peptide and one copy of a second MGS peptide that is different from the first MGS peptide. Embodiments include other multimers, such as trimers, pentamers, hexamers, and heptamers, among others.

[0131] As further described herein, in some embodiments, a linker structure comprising multiple linkers can conjugate at least two MGS peptides together to form the multimer. Example multimers and linker structures are illustrated in connection with at least FIGs. IB- IE.

[0132] In some embodiments, the MGS peptide has a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences set forth in SEQ ID NOs: 1-60, such as any sequences set forth in sequences 1-58. In some embodiments, the MGS peptide 100 has 100% identity in the active portion of the peptide, wherein the active portion is the portion that retains its ability to target lung fibroblasts. Thus, in some embodiments, the at least 90%, 91%, 92%, 93%,PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)94%, 95%, 96%, 97%, 98%, or 99% identity with any of the MGS peptides occurs outside of the active portion.

[0133] In some embodiments, the MGS peptide can be or is modified to optimize and / or stabilize the MGS peptide. Optimized MGS peptides can be obtained by applying modifications to the individual parental peptide sequences. These modifications can be used to identify the amino acids within the parental sequence that are required for specific binding and internalization to lung fibroblasts. These modifications can be obtained by a combination of alanine scanning and truncations of the amino-terminal region and C-terminal region of the parental peptide. PEG can provide protection of the C-terminus of the MGS peptide, provide a spacer between the peptide and the cargo attached through the amino acid at the C-terminus, and enhance solubility of the MGS peptide. Modification at the N-terminus by acetylation (CH3CO-) or other acylation, cyclization, amino-alkylation, methylation, succinyl ati on, pegylation, and / or d-amino acids, such as d(L) can protect against degradation. There is not a uniform length of optimized peptide that can be applied to all MGS peptides and all changes can be tested to confirm the effect on peptide uptake and stability. Thus, in some embodiments, the MGS peptide has an N-terminal protecting group, such as those described above. For example, as shown by Table 1, the MGS peptides disclosed herein are modified by acetylation on the N-terminus. Accordingly, in some embodiments, the N-terminal protecting group comprises an acetyl group (Ac = CH3CO). In some embodiments, the N-terminal protecting group can be, but is not limited to, an acyl group, a cyclic group, an amin-alkyl group, a succinyl group, a PEG group, Formyl, methyl, CH3-(CH)n-CO, Fluorophore, Fatty acid, alkyl amine, aryl groups, carbohydrates, sulfonamide, or carbamate, among other groups such as those illustrated and described above. As may be appreciated, “Ac” is sometimes herein interchangeably used to refer to acetylation or an acetyl group (CH3CO). In some embodiments, the MGS peptide can be or is modified by adding amino acids or replacing an amino acid(s) to the individual parental peptide sequences. For example, as least one amino acid of the parental peptide sequence can be replaced with another (such as another amino acid, a modified amino acid, or an isomer, such as Norleucine (Nle)). In some embodiments or in addition, asPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) least one amino acid can be added to the parental peptide sequence, such as the addition of amino acid(s) within the sequence.

[0134] As noted above and further described herein, the MGS peptides of various embodiments are capable of carrying cargo to and mediating internalization of the cargo to the lung fibroblasts. In some embodiments, multimer forms of the MGS peptides (e.g., dimers, trimers, or tetramers) show greater internalization in lung fibroblasts than monomeric forms. Additionally, the MGS peptides are capable of carrying and mediating internalization of a variety of different cargos.

[0135] The MGS peptides disclosed herein are unique in enabling internalization (e.g., uptake) of a variety of potential therapeutics and other cargo by lung fibroblasts. Such MGS peptides are selected for their targeting and internalization by the specific cell type of lung fibroblasts. In turn, the cell specific uptake can assist in concentrating the therapeutic in the correct cell type and potentially decreasing off-target effects by preventing uptake in other cell types. Small molecule drugs typically enter all cells, which can result in a variety of off-target effects that limit the dose or use of the potential therapy. The MGS peptides can be used for cell-specific therapy and with the use of nucleic acid cargo or other biologic macromolecules for intracellular therapies, such as for therapeutic intervention in pulmonary fibrosis.

[0136] In some embodiments, the MGS peptides disclosed herein can be used for therapy to block the progression of pulmonary fibrosis. These effects can depend on the state of the lungs at diagnosis. The effect in early-stage disease is the retention of lung capacity (e.g., tidal volume of air that enters the lung for gas exchange), pliability of lung tissue (e.g., lack of stiffening), retention of air sac structure with the lung, longer survival, and reduced need for lung transplantation. The air sacs structure can be required for the efficient exchange of gases between the blood and air within the lung.

[0137] FIGs. 1 A-1E illustrate example MGS compounds and MGS-cargo conjugates comprising an MGS peptide, in accordance with the present disclosure. In some embodiments, the MGS compounds and MGS-cargo conjugates 110, 120, 130, 140, 160 of FIGs. 1 A-1E can include at least one MGS peptide as previously described.

[0138] As further described herein, FIG. 1 A depicts an MGS compound 110 with multiple MGS peptides 100A, 100B. . . 100N conjugated to a (common) linker 112 and aPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) reactive group 114 for binding to a cargo. FIG. IB depicts an MGS-cargo conjugate 120 with a single MGS peptide 100 conjugated to a cargo 122, directly or indirectly through an optional linker 124. FIG. 1C depicts an example linker structure 141 for an MGS compound 130 with multiple MGS peptides 100A, 100B. . . 100N. FIG. ID depicts an MGS compound 140 comprising two MGS peptides 100A, 100B conjugated to a linker structure 142 where the linker structure 142 comprises multiple linkers 144A, 144B, 146, 148 including PEG linkers 144A, 144B conjugated to the MGS peptides 100A, 100B and to a branch linker 146 and the branch linker 146 is conjugated to a reactive group 148 for attachment of cargo or other moieties. FIG. IE depicts an MGS compound 160 comprising four MGS peptides 100A, 100B, 100C, 100D conjugated to a linker structure 162 where the linker structure 162 comprises multiple linkers 144A, 144B, 144C, 144D, 146A, 146B, 146C, 148 including PEG linkers 144A, 144B, 144C, 144D conjugated to the MGS peptides 100A, 100B, 100C, 100D and also to branch linkers 146A, 146B, 146C and the branch linkers 146A, 146B, 146C conjugated to each other and to a reactive group 148 for attachment of cargo or other moieties.

[0139] FIG. 1 A illustrates an example of an MGS compound 110 that comprises a multimer. For example, the MGS compound 110 comprises at least two MGS peptidesIOO A, 100B. . . 100N. In some embodiments, the at least two MGS peptides 100 A,IOOB. . . 100N are each independently selected from SEQ ID NOs: 1-60, SEQ ID NOs: 1-58, SEQ ID NOs: 1-29, SEQ ID NOs: 1-2, 7-8, 15, 19, and 23-29, SEQ ID NOs: 1-2, 7-8, 15, 19, and 23-29, or SEQ ID NOs: 1-2, 7-8, 15, 19, 24, and 27-28, among other combinations. In some embodiments, each MGS peptide 100A, 100B. . . 100N independently comprises a sequence of SEQ ID NO: 59 or 60.

[0140] In some embodiments, the at least two MGS peptides 100A, 100B. . . 100N comprise two MGS peptides 100A, 100B, such as further illustrated and described by the dimer of FIG. ID. In some embodiments, the at least two MGS peptides 100A, 100B comprise four MGS peptides, such as further illustrated and described by the tetramer of FIG. IE.

[0141] In some embodiments, the at least two MGS peptides 100A, 100B. . . 100N are each the same MGS peptide sequence (e.g., multiple copies of the same MGS peptide). In other embodiments, at least two of the at least two MGS peptides 100A,PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)100B. . . 100N are different from one another. In some embodiments, each (e.g., all) of the at least two MGS peptides 100 A, 100B. . . 100N are different from one another.

[0142] In some embodiments, the at least two MGS peptides 100A, 100B. . . 100N can include protecting groups. For example, the at least two MGS peptides 100 A, 100B. . . 100N can each comprise a protecting group on the N-terminus, the protecting groups each being independently selected from an acyl group, a cyclic group, an aminoalkyl group, a succinyl group, a PEG group, a methyl group, and a combination thereof. In some such embodiments, the at least two MGS peptides 100 A, 100B. . . 100N are each independently selected from SEQ ID NOs: 1-29 or from SEQ ID NOs: 59-60.

[0143] In some embodiments, the MGS compound 110 further comprises at least one linker 112. The linker 112 can comprise a PEG linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

[0144] In some embodiments, the linker 112 comprises multiple linkers forming a linker structure, as further illustrated by FIGs. 1C-1E. For example, the linker structure can include at least two PEG linkers, each respectively conjugated to one of the at least two MGS peptides 100 A, 100B. . . 100N, directly or indirectly, and at least one branch linker conjugated to two of the at least two PEG linkers, directly or indirectly. As further described herein, branch linker can comprise a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid. In some embodiments, as further described herein, the linker structure further comprises additional linkers comprising reactive groups and the at least two PEG linkers are indirectly conjugated to, respectively, the one of the at least two MGS peptides 100A, 100B. . . 100N through the reactive groups.

[0145] In some embodiments, the MGS compound 110 further comprises a reactive group 114 conjugated to the linker 112, directly or indirectly, and to allow for conjugation to a cargo, such as the cargo 122 illustrated by FIG. IB. Example reactive groups include carboxylic acids, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (with No Acetylenic Hydrogen), amides, imides, amines, thiols, phosphines, pyridines, anhydrides, azo compounds, diazo compounds, azido compounds, hydrazine, azide compounds, carbamates, epoxides, esters, sulfate esters,PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) phosphate, thiophosphate esters, borate esters, halogenated organic compounds, isocyanates, isothiocyanates, ketones, oximes, sulfides (Organic), lipids, hydrogen, and combinations thereof, among other reactive moieties.

[0146] In some embodiments, the MGS compound 110 comprises the structure ofwherein each X comprises one of the at least two MGS peptides 100A, 100B. . . 100N, and Z and Z’ comprise reactive groups (e.g., 114). As further described herein, Z and / or Z’ can be configured to react with cargo. In some embodiments, the above structure can include a dimer 140 of FIG. ID.

[0147] In some embodiments, the MGS compound 110 comprises the structure ofwherein each X comprises one of the at least two MGS peptides 100A, 100B. . . 100N, and Z and Z’ comprise reactive groups (e.g., 114). In some embodiments, Z and / or Z’ can be configured to react with cargo. In some embodiments, the above structure can include a tetramer 160 of FIG. IE.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0148] In some embodiments, Z and Z’ can each be independently selected from carboxylic acids, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (with No Acetylenic Hydrogen), amides, imides, amines, thiols, phosphines, pyridines, anhydrides, azo compounds, diazo compounds, azido compounds, hydrazine, azide compounds, carbamates, epoxides, esters, sulfate esters, phosphate, thiophosphate esters, borate esters, halogenated organic compounds, isocyanates, isothiocyanates, ketones, oximes, sulfides (Organic), lipids, hydrogen, and combinations thereof, among other reactive moieties.

[0149] FIG. IB illustrates an example MGS-cargo conjugate 120 comprising an MGS peptide 100 conjugated to a cargo 122, directly or indirectly through a linker 124. The MGS peptide 100 can include an implementation of and / or at least some of the same features and attributes as the MGS peptides as previously described. In some embodiments, the MGS peptide 100 is selected from SEQ ID NOs: 1-60. In some embodiments, the MGS peptide 100 is selected from SEQ ID NOs: 1-60, SEQ ID NOs: 1-58, SEQ ID NOs: 1-29, SEQ ID NOs: 1-2, 7-8, 10, 15, 19, 21, and 23-29, or SEQ ID NOs: 1-2, 7-8, 10, 15, 19, 21, 24, and 27-28, among other combinations. In some embodiments, the MGS peptide 100 comprises the sequence of SEQ ID NO: 59 or 60.

[0150] In some embodiments, as previously described, the MGS peptide 100 can include a protecting group. For example, the MGS peptide 100 can comprise any of SEQ ID NOs: 1-29 and further comprises a protecting group on the N-terminus selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a PEG group, a methyl group, and a combination thereof.

[0151] Example cargo 122 includes a nucleic acid, a peptide, a protein, an antibody, a lipid, an imaging agent, a dye, a therapeutic, a small molecule, a radionuclide, a carbohydrate, and a nanoparticle, among other molecules and compounds and combinations thereof. In some embodiments, the cargo 122 includes a nucleic acid selected from ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and a combination thereof. In some embodiments, the cargo 122 includes a small interfering ribonucleic acid (siRNA), among other types of RNA and / or DNA sequences.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0152] In some embodiments, the MGS peptide 100 can selectively and / or specifically bind to a lung fibroblast and internalize the cargo 122 into the lung fibroblast.

[0153] In some embodiments, the MGS-cargo conjugate 120 can comprise multimers, such as further illustrated by MGS compounds of FIGs. 1 A and 1C-1E. For example, the MGS-cargo conjugate 120 can comprise two MGS peptides including a first MGS peptide 100 and a second MGS peptide (not illustrated by FIG. IB). In some such embodiments, the MGS-cargo conjugate 120 can be referred to as a dimer. In some embodiments, the two MGS peptides are the same MGS peptide (e.g., two copies of the same MGS peptide). In other embodiments, the two MGS peptides are different from one another. In some embodiments, the two MGS peptides are indirectly conjugated through a linker structure, as further described herein.

[0154] In some embodiments, the MGS-cargo conjugate 120 can comprise four MGS peptides including a first MGS peptide 100, a second MGS peptide, a third MGS peptide, and a fourth MGS peptide (not illustrated by FIG. IB). In some such embodiments, the MGS-cargo conjugate 120 can be referred to as a tetramer. In some embodiments, the four MGS peptides are the same MGS peptide (e.g., four copies of the same MGS peptide). In other embodiments, at least two of the four MGS peptides are different from one another. For example, two, three, or all four of the MGS peptides can be different from one another. In some embodiments, the four MGS peptides are conjugated through a linker structure, as further described herein.

[0155] In various embodiments, linkers can conjugate the at least two MGS peptides together, conjugate respective linkers together, or conjugate a linker or MGS peptide to a cargo. In some embodiments, the linker structure can be used to conjugate the at least two MGS peptides and at least a cargo 122 together. The linker structure can comprise a plurality of linkers, such as but not limited to, PEG linker(s), branch linker(s), and at least one reactive group. As previously described, example linkers include a PEG linker, an alkyl linker, a maleimide linker, an amino acid linker, an amide linker, a thiol linker, an amine linker, an aryl linker, and a reactive group, among others.

[0156] In some embodiments, the MGS-cargo conjugate 120 comprises a dimer comprising the first MGS peptide 100 and the second MGS peptide. The first MGSPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) peptide 100 and the second MGS peptide can be each independently selected from SEQ ID NOs: 1-60, SEQ ID NOs: 1-58, SEQ ID NOs: 1-29, SEQ ID NOs: 1-2, 7-8, 15,19, and 23-29, or SEQ ID NOs: 1-2, 7-8, 15, 19, 24, and 27-28, among other combinations. In some embodiments, the first and second MGS peptide each independently comprise a sequence of SEQ ID NOs: 59 or 60.

[0157] In some embodiments, the dimer further comprises a linker 124. The linker 124 can include any of the above-described linkers and combinations thereof. In some embodiments, the dimer comprises a PEG linker on the C-terminus of each of the first MGS peptide 100 and the second MGS peptide and a lysine branch linker that links the PEG linkers.

[0158] In some embodiments, the dimer comprises a linker structure, which can include the linker 124. The linker structure can comprise a first PEG linker conjugated to the first MGS peptide 100 (directly or indirectly), a second PEG linker attached to the second MGS peptide (directly or indirectly), and a branch linker conjugated to the first PEG linker and to the second PEG linker together, directly or indirectly. In such embodiments, the branch linker can indirectly link the first and second MGS peptides together, and the directly or indirectly link the first PEG linker and the second PEG linker. In such embodiments, the first PEG linker can be indirectly conjugated to the second PEG linker through the branch linker. In some embodiments, the first and second PEG linkers comprise PEGn or PEG12, although embodiments can include other numbers of PEG units. As previously described, the PEG linkers can be a sufficient length to separate the first MGS peptide 100 from the cargo 122 to prevent any steric interference between the first MGS peptide 100 (and the second MGS peptide) and the cargo 122. A branch linker, as used herein, refers to or includes a linker that connects MGS chains which comprise at least an MGS peptide and a linker, such as a PEG linker that connects the MGS peptide to the branch linker.

[0159] The branch linker can be conjugated to the first MGS peptide 100 and to the second MGS peptide, directly or indirectly, such that the branch linker links the first and second MGS peptides. In some embodiments, the branch linker can include a modified amino acid. For example, the branch linker can be a functionalized lysine, aPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) functionalized cysteine, a functionalized glutamic acid, or a functionalized aspartic acid, among other modified amino acids.

[0160] In some embodiments, the linker structure further comprises a reactive group conjugated to the branch linker and conjugated to the cargo 122. The reactive group can include any of the previously described reactive groups. In various embodiments, the reactive group is conjugated to the C-terminal side of the branch linker which comprises an amino acid.

[0161] In some embodiments, the linker structure of the dimer can comprise additional linkers, such as additional reactive groups and as further illustrated in connection with FIG. ID. In some embodiments, the first PEG linker and the second PEG linker are indirectly conjugated to, respectively, the first MGS peptide 100 and the second MGS peptide through reactive groups, such as -NH or -CONH.

[0162] In some embodiments, the linker structure of a dimer can comprise:wherein Z and Z’ comprise reactive groups. In some embodiments, the Z and / or Z’ reactive groups can be configured to react with a cargo, as further described herein.

[0163] In some embodiments, the MGS-cargo conjugate 120 comprises a tetramer comprising a first MGS peptide 100, a second MGS peptide, a third MGS peptide, and a fourth MGS peptide. The first, second, third, and fourth MGS peptides can be each independently selected from SEQ ID NOs: 1-60, SEQ ID NOs: 1-58, SEQ ID NOs: 1-29, SEQ ID NOs: 1-2, 7-8, 15, 19, and 23-29, or SEQ ID NOs: 1-2, 7-8, 15, 19, 24, and 27-28, among other combinations. In some embodiments, the four MGS peptides each independently comprise a sequence of SEQ ID NOs: 59 or 60. In some embodiments, the tetramer further comprises a linker 124. The linker 124 can include any of the above described linkers and combinations thereof. In some embodiments, the tetramer comprises a PEG linker on the C-terminus of each of the MGS peptide 100, thePCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) second MGS peptide, the third MGS peptide, and the fourth MGS peptides, and lysine branch linkers that link the PEG linkers.

[0164] In some embodiments, the tetramer comprises a linker structure, which can include the linker 124. The linker structure can comprise a first PEG linker conjugated to the MGS peptide 100 (directly or indirectly), a second PEG linker conjugated to the second MGS peptide (directly or indirectly), a third PEG linker conjugated to the third MGS peptide (directly or indirectly), and a fourth PEG linker conjugated to the fourth MGS peptide (directly or indirectly). The linker structure can further comprise branch linkers that are respectively conjugated, directly or indirectly: (i) to the first PEG linker and to the second PEG linker, (ii) to the second PEG linker and to the third PEG linker, and (iii) to the third PEG linker and to the fourth PEG linker. In such embodiments, the branch linkers can indirectly link the first, second, third, and fourth MGS peptides together, and directly or indirectly link the first PEG linker and the second PEG linker, the second PEG linker and the third PEG linker, and the third PEG linker and the fourth PEG linker. In some embodiments, the first, second, third, and fourth PEG linkers can be indirectly conjugated to each other via the branch linkers and additional linkers, such as additional PEG linkers and amino acid linker and / or other reactive groups, as further described herein. In some embodiments, the first, second, third, and fourth PEG linkers (each) comprise PEGn or PEG12, although embodiments can include other numbers of PEG units.

[0165] In some embodiments, the branch linker can include a modified amino acid. For example, the branch linker can be a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, or a functionalized aspartic acid.

[0166] In some embodiments, the linker structure further comprises a reactive group conjugated to at least one of the branch linkers and conjugated to the cargo 122. The reactive group can include any of the previously described reactive groups. In various embodiments, the reactive group conjugated to the C-terminal side of the branch linker, wherein the branch linkers comprise amino acids.

[0167] In some embodiments, the linker structure of the tetramer can comprise additional linkers, such as additional PEG linkers and reactive groups and as further illustrated in connection with FIG. IE. In some embodiments, the first PEG linker,PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) second PEG linker, the third PEG linker, and the fourth PEG linker are indirectly conjugated to, respectively, the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptide through the reactive groups, such as -CONH.

[0168] In some embodiments, the linker structure of a tetramer can comprise:wherein Z and Z’ comprise reactive groups. In some embodiments, Z and / or Z’ are reactive groups configured to react with a cargo.

[0169] In some embodiments, the MGS-cargo conjugate 120 comprises the structure of:wherein each X comprises the MGS peptide, and Z and Z’ comprise reactive groups. At least one of the Z and Z’ reactive groups can be configured to react with a cargo. In some embodiments, the above illustrated structure can be referred to as a “dimer core”.

[0170] In some embodiments, the MGS-cargo conjugate 120 comprises the structure of:PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)wherein each X comprises the MGS peptide, and Z and Z’ comprise reactive groups. In some embodiments, at least one of the Z and Z’ reactive groups can be configured to react with a cargo. In some embodiments, the above illustrated structure can be referred to as a “tetramer core”.

[0171] In some embodiments, Z and Z’ can include any of the above-described reactive groups, which are not repeated for ease of reference.

[0172] In various embodiments, Z and / or Z’ is configured to react with and are conjugated to the cargo 122 or a portion thereof. In some embodiments, one of Z and Z’ is conjugated to the cargo 122 and the other of Z and Z’ is H, which may not be reacted with a cargo. Embodiments are not so limited and the other of Z and Z’ which is not conjugated to a cargo can be conjugated to another type of moiety. In some embodiments, the cargo 122 includes a first cargo and a second cargo, and Z is conjugated to the first cargo and Z’ is conjugated to the second cargo. The first cargo and second cargo can include the same type or different type of cargos.

[0173] FIG. 1C illustrates an example of a linker structure 141 of an MGS compound 130. As shown, the MGS compound 130 comprises at least two MGS peptides 100A, 100B. . . 100N, as previously described in connection with FIG. 1 A, the common features not being repeated for ease of reference and as illustrated by the common reference numerals.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0174] As shown by FIG. 1C, the linker structure 141 comprises chains 145A, 145B ....145N of linkers which are each directly and / or indirectly conjugated to a respective one of the at least two MGS peptides 100 A, 100B. . . 100N and to branch linker(s) 146A. . . 146N-1 which conjugate the at least two MGS peptides 100A, 100B. . . 100N together. At least one branch linker 146A. . . 146N-1 is conjugated to a reactive group 148 which can react with and conjugate to a cargo. The chains 145A, 145B. . . .145N of linkers comprise at least PEG linkers 144A, 144B. . . 144N which are between the respective MGS peptide 100 A, 100B. . . 100N and the branch linker 146A. . . 146N-1. In some embodiments, the PEG linkers 144A, 144B. . . 144N comprise between one PEG unit and 24 PEG units or more, which is sometimes herein referred to as PEG1-PEG24. The PEG linkers 144A, 144B. . . 144N between the MGS peptides 100A, 100B. . . 100N and the branch linker(s) 146A. . . 146N-1 provide solubility and separate the MGS peptides 100 A, 100B. . . 100N from the cargo(s) to prevent steric interference between the MGS peptides 100A, 100B. . . 100N and the cargo(s).

[0175] As previously described and further illustrated by the MGS compounds 140, 160 of FIGs. 1D-1E, the linker structure 141 can include additional linkers. For example, the chains 145 A, 145B. . . 145N of linkers can include reactive groups which are directly conjugated to the MGS peptides 100A, 100B. . . 100N and / or the branch linker(s) 146A. . . 146N-1. In some embodiments, the chains 145A, 145B. . . 145N of linkers can include additional PEG linkers and / or reactive groups between the PEG linkers 144A, 144B. . . 144N and the branch linker(s) 146A. . . 146N-1. In some embodiments, the linker structure 141 can include additional reactive groups conjugated to at least one of branch linker(s) 146A. . . 146N-1, such that multiple cargos may be conjugated to the linker structure 141.

[0176] The reactive group 148 can react with the cargo using a variety of techniques. For example, the cargo can react with the reactive group 148 using amide chemistry, maleimide chemistry, click chemistry, and / or hydrozone linkers, among other techniques. In some embodiments, the linker structure 141 includes multiple reactive groups conjugated to the branch linker, and one reactive group may be reacted with and conjugated to cargo and the other may be reactive with and conjugated to other cargo, such as a lipid, or may not be further reacted.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0177] FIG. ID illustrates an example MGS compound 140 comprising a dimer that includes two MGS peptides 100A, 100B, herein generally referred to as “dimer 140” for ease of reference. In some embodiments, MGS peptides 100A, 100B are each individually selected from SEQ ID NOs: 1-60, SEQ ID NOs: 1-58, SEQ ID NOs: 1-29, SEQ ID NOs: 1-2, 7-8, 15, 19, and 23-29, or SEQ ID NOs: 1-2, 7-8, 15, 19, 24, and 27- 28, as previously described. In some embodiments, the two MGS peptides 100 A, 100B each independently comprise a sequence of SEQ ID NOs: 59 or 60.

[0178] In some embodiments, the dimer 140 comprises a linker structure 142. In some embodiments, the linker structure 142 comprises a first PEG linker 144A conjugated to a first MGS peptide 100 A of the two MGS peptides 100 A, 100B (directly or indirectly), a second PEG linker 144B conjugated to a second MGS peptide 100B of the two MGS peptides 100 A, 100B (directly or indirectly), and a branch linker 146 conjugated to the first PEG linker 144 A and to the second PEG linker 144B (directly or indirectly). The first and second PEG linkers 144 A, 144B can comprise PEGn or PEG12, among other numbers of PEG units. In some embodiments, dimer 140 comprises a PEG linker 144 A, 144B on the C-terminus of each of two MGS peptides 100 A, 100B and a lysine branch linker (e.g., branch linker 146) conjugated to the PEG linkers 144A, 144B.

[0179] In some embodiments, the branch linker 146 comprises an amino acid. For example, the branch linker 146 can comprise a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0180] In some embodiments, the linker structure 142 further comprises a reactive group 148 conjugated to the branch linker 146, directly or indirectly, and to allow for conjugation to a cargo, such as the cargo 122 illustrated by FIG. IB. For example, the reactive group 148 can be conjugated on the C-terminal side of the branch linker 146 comprising an amino acid.

[0181] In some embodiments, the linker structure 142 can include additional linkers. For example, the additional linkers can include reactive groups 143 A, 143B between the MGS peptides 100 A, 100B and the PEG linkers 144 A, 144B. In some embodiments, the reactive groups 143 A, 143B between the MGS peptides 100A, 100B and the PEGPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) linkers 144 A, 144B can include -NH or -CONH, but examples are not so limited and other reactive groups can be used. In some such embodiments, the first PEG linker 144 A and the second PEG linker 144B are indirectly conjugated to, respectively, the first MGS peptide 100 A and the second MGS peptide 100B through the reactive groups 143A, 143B.

[0182] In some embodiments, the linker structure 142 of the dimer 140 comprises:wherein Z and Z’ comprise reactive groups.

[0183] In some embodiments, at least one of Z and Z’ is configured to react with a cargo or a portion thereof. In some embodiments, one of Z and Z’ is configured to react the with cargo, and the other of Z and Z’ is H. In some embodiments, Z’ is configured to react with a first cargo and Z’ is configured to react with a second cargo, wherein the first cargo and the second cargo are the same or different (e.g., different types of cargos). For example, the first cargo can include a lipid and the second cargo can include a nucleic acid, among other combinations. In some embodiments, one of Z and Z’ is a lipid, although embodiments are not so limited.

[0184] As previously described, the cargo can be, but is not limited to, a dye, an imaging agent, a therapeutic, a protein, a nucleic acid, an amino acid, a peptide, a lipid, an antibody, a small molecule, a radionuclide, carbohydrate, or a nanoparticle.

[0185] FIG. IE illustrates an example MGS compound 160 comprising a tetramer that includes four MGS peptides 100 A, 100B, 100C, 100D, herein generally referred to as “tetramer 160” for ease of reference. In some embodiments, MGS peptide 100A, 100B, 100C, 100D are each individually selected from SEQ ID NOs: 1-60, SEQ ID NOs: 1-58, SEQ ID NOs: 1-29, SEQ ID NOs: 1-2, 7-8, 15, 19, and 23-29, or SEQ ID NOs: 1-2, 7-8,PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)15, 19, 24, and 27-28, as previously described. In some embodiments, each MGS peptide 100A, 100B, 100C, 100D independently comprises SEQ ID NOs: 59 or 60.

[0186] In some embodiments, the tetramer 160 comprises a linker structure 162. In some embodiments, the linker structure 142 comprises a first PEG linker 144A conjugated to a first MGS peptide 100A of the four MGS peptides 100A, 100B, 100C, 100D (directly or indirectly), a second PEG linker 144B conjugated to a second MGS peptide 100B of the four MGS peptides 100 A, 100B, 100C, 100D (directly or indirectly), a third PEG linker 144C conjugated to a third MGS peptide 100C of the four MGS peptides 100 A, 100B, 100C, 100D (directly or indirectly), and a fourth PEG linker 144D conjugated to a fourth MGS peptide 100D of the four MGS peptides 100A, 100B, 100C, 100D (directly or indirectly). The linker structure 162 can further comprise branch linkers 146 A, 146B, 146C that respectively are conjugated (directly or indirectly): (i) to the first PEG linker 144 A and to the second PEG linker 144B, (ii) to the second PEG linker 144B and to the third PEG linker 144C, and (iii) to the third PEG linker 144C and to the fourth PEG linker 144D. The first, second, third, and fourth PEG linkers 144A, 144B, 144C, 144D can comprise PEGn or PEG12, among other numbers of PEG units. In some embodiments, tetramer 160 comprises a PEG linker 144A, 144B, 144C, 144D on the C-terminus of each of the four MGS peptides 100A, 100B, 100C, 100D and lysine branch linkers (e.g., 146A, 146B, 146C) conjugated to the respective PEG linkers 144A, 144B, 144C, 144D, directly or indirectly through additional linkers.

[0187] In some embodiments, the branch linkers 146A, 146B, 146C can each comprise an amino acid. For example, each of the branch linkers 146A, 146B, 146C can comprise a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0188] In some embodiments, the linker structure 162 further comprises a reactive group 148 conjugated to at least one of branch linkers 146A, 146B, 146C, directly or indirectly, and to allow for conjugation to a cargo, such as the cargo 122 illustrated by FIG. IB. For example, at least one reactive group 148 can be on the C-terminal side of the branch linker 146B comprising an amino acid.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0189] In some embodiments, the linker structure 162 can include additional linkers, such as linkers between the PEG linkers 144A, 144B, 144C, 144D on the C-terminal side of each of the four MGS peptides 100 A, 100B, 100C, 100D and the branch linkers 146A, 146B, 146C. In some embodiments, the additional linkers can include modified cysteine and mal eimide linkers (e.g., cysteine-maleimide linkers) and additional PEG linkers. In some embodiments, each of the additional PEG linkers can include the same length or different length of PEG units as the PEG linkers. In such embodiments, the linker structure 162 comprises, for each MGS peptide 100A, 100B, 100C, 100D, at least two PEG linkers and a reactive group (e.g., cysteine- maleimide linker) between the at least two PEG linkers, with the reactive groups connecting the at least two PEG linkers. For example, the additional PEG linkers can each include PEGn or PEG12 in some embodiments.

[0190] Accordingly, in some embodiments, the linker structure 162 comprises additional PEG linkers and reactive groups, wherein the branch linkers 146 A, 146B, 146C are indirectly conjugated to the first PEG linker 144 A and to the second PEG linker 144B, to the second PEG linker 144B and to the third PEG linker 144C, and to the third PEG linker 144C and to the fourth PEG linker 144D through the additional PEG linkers and reactive groups.

[0191] In some such embodiments, the linker structure 162 further comprises a first reactive group (e.g., cysteine-maleimide linker) and a fifth PEG linker, the first reactive group being between the first PEG linker 144 A linker and the fifth PEG linker, and a first of the branch linkers 146A being indirectly conjugated to the first PEG linker 144A through the first reactive group (e.g., cysteine-maleimide linker) and the fifth PEG linker. The linker structure 162 can further comprise a second reactive group (e.g., cysteine-maleimide linker) and a sixth PEG linker, the second reactive group (e.g., cysteine-maleimide linker) being between the second PEG linker 144B and the sixth PEG linker, and the first and a second of the branch linkers 146 A, 146B being indirectly conjugated to the second PEG linker 144B through the second reactive group (e.g., cysteine-maleimide linker) and the sixth PEG linker. The linker structure 162 can further comprise a third reactive group (e.g., cysteine-maleimide linker) and a seventh PEG linker, the third reactive group (e.g., cysteine-maleimide linker) being between thePCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) third PEG linker 144C and seventh PEG linker, and the second and a third of the branch linkers 146B, 146C being indirectly conjugated to the third PEG linker 144C through the third reactive group (e.g., cysteine-maleimide linker) and the seventh PEG linker. The linker structure 162 can further comprise a fourth reactive group (e.g., cysteine- maleimide linker) and eighth PEG linker, the fourth reactive group (e.g., cysteine- maleimide linker) being between the fourth PEG linker 144D and eighth PEG linker, and the third of the branch linkers 146C being indirectly conjugated to the fourth PEG linker 144D through the fourth reactive group (e.g., cysteine-maleimide linker) and the eighth PEG linker.

[0192] In some embodiments, the additional linkers can include reactive groups 143 A, 143B, 143C, 143D between the four MGS peptides 100A, 100B, 100C, lOOD and the PEG linkers 144A, 144B, 144C, 144D. In some embodiments, the reactive groups 143 A, 143B, 143C, 143D between the four MGS peptides 100A, 100B, 100C, 100D and the PEG linkers 144A, 144B, 144C, 144D can include -CONH, but examples are not so limited and other reactive groups can be used. In some such embodiments, the first PEG linker 144A, the second PEG linker 144B, the third PEG linker 144C, and the fourth PEG linker 144D are indirectly conjugated to, respectively, the first MGS peptide 100 A, the second MGS peptide 100B, the third MGS peptide 100C, and the fourth MGS peptide 100D through the reactive groups 143A, 143B, 143C, 143D.

[0193] In some embodiments, the linker structure 162 of the tetramer 160 comprises:PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) wherein Z and Z’ comprises reactive groups.

[0194] In some embodiments, the Z and / or Z’ reactive groups can be configured to react with a cargo or a portion thereof. As previously described, a cargo can be, but is not limited to, a dye, an imaging agent, a therapeutic, a protein, a nucleic acid, an amino acid, a peptide, a lipid, an antibody, a radionuclide, carbohydrate or a nanoparticle. In some embodiments, one of Z and Z’ is conjugated to a cargo and the other of Z and Z’ is H, which may not be further reacted with a cargo. In some embodiments, both of Z and Z’ are independently conjugated to cargo, which may be the same or different cargo. For example, the first cargo may include a lipid and the second cargo may include a nucleic acid, such as RNA, among other combinations. In some embodiments, one of Z and Z’ is a lipid, although embodiments are not so limited.

[0195] In some embodiments, the MGS compound 140 of FIG. ID including the linker structure 142 used to conjugate the two MGS peptides 100A, 100B together and the MGS compound 160 of FIG. IE including the linker structure 162 used to conjugate the four MGS peptides 100 A, 100B, 100C, 100D together may be respectively referred to as a “dimer core” (if linking two MGS peptides) and a “tetramer core” (if linking four MGS peptides). The linker structures 142, 162 are examples of linkers used in dimer and tetramer cores. In other embodiments, PEG linkers of other lengths can be used. For example, PEG linkers of between 1-5000 PEG units can be used.

[0196] In some embodiments, linker structures 142, 162 comprise at least one reactive group configured to react with and conjugate to a C-terminus of an MGS peptide and at least one additional reactive group configured to react with and conjugate to a cargo. The reactive group configured to conjugate to a C-terminus of the MGS peptide can include -NH or -CONH, among other groups. For example, the dimer core can include an -NH or a -CONH group directly attached to the C-terminus of each MGS peptide and which are directly attached to the PEG linkers. For the tetramer core, in some embodiments, an -NH or a -CONH can be directly attached to the C-terminus of each MGS peptide and which are directly attached to the (first) PEG linkers. In some embodiments, the dimer core can be linear. In some embodiments, the tetramer core can be non-linear.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0197] As previously described, the linker structures 142, 162 can include multiple linkers which respectively link at least two MGS peptides and the at least two MGS peptides to a cargo. In some embodiments, a PEG linker can be used to conjugate an MGS peptide to the cargo, directly or indirectly. In some embodiments, the linker between the MGS peptide and the cargo can be longer than the linker between two MGS peptides.

[0198] In some embodiments, a linker structure 142, 162 comprises at least two PEG linkers and a reactive group between at least two of the PEG linkers. In some embodiments, the reactive group connects at least two PEG linkers.

[0199] Example linkers of linker structures 142, 162 include an amino acid, a peptide, an alkyl, a maleimide, an amino acid-maleimide linker, a thiol, hydrazone, amide, and a reactive group, among other groups. In some embodiments, the amino acid can be a modified amino acid. For example, a modified amino acid can be a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, or a functionalized aspartic acid, among other modified amino acids. In some embodiments, a linker of the linker structure 142, 162 comprises biotin.

[0200] Embodiments are not limited to the MGS peptides of SEQ ID NOs: 1-60. Further, MGS compounds are not limited to multimers and can include single MGS peptides.

[0201] Various embodiments are directed to methods of using any of the above described MGS peptides, MGS compounds, and MGS-cargo conjugates.

[0202] In the methods described herein, delivery (or administration) of the MGS peptides, MGS compounds, or MGS-cargo conjugates disclosed herein can be via a variety of mechanisms. As defined above, disclosed herein are MGS compounds and / or MGS-cargo conjugates comprising an MGS peptide described herein can be used to produce a composition which can also include a carrier such as a pharmaceutically acceptable carrier. For example, disclosed are pharmaceutical compositions, comprising the MGS peptides disclosed herein, and a pharmaceutically acceptable carrier.

[0203] For example, the compositions described herein can comprise a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material or carrier that would be selected to minimize any degradation of the activePCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Examples of carriers include dimyristoylphosphatidyl choline (DMPC), phosphate buffered saline or a multivesicular liposome. For example, PG:PC:Cholesterol:peptide or PC:peptide can be used as carriers in this invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Other examples of the pharmaceutically acceptable carrier include, but are not limited to, saline, Ringer’s solution and dextrose solution. The pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0204] Pharmaceutical compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the polypeptide, peptide, nucleic acid, vector of the invention is not compromised. Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, and anesthetics, among others. The pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

[0205] Preparations of parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueousPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0206] Formulations for optical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0207] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions can potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0208] The disclosed delivery techniques can be used not only for the disclosed compositions but also the disclosed nucleic acid sequences and vectors.

[0209] Various methods of the present disclosure are directed to transporting a cargo to the lung fibroblasts of a subject by administering a composition disclosed herein to a subject in need thereof. An example method for targeting lung fibroblasts in a subject comprises administering any of the above-described MGS compounds and MGS-cargo conjugates to the subject, such as a subject diagnosed with or having pulmonary fibrosis or emphysema or which is genetically disposed to having pulmonary fibrosis or emphysema. In some embodiments, administering is an intravenous, intrathecal, subcutaneous, intramuscular, intraperitoneal, intradermal, or intracardiac administration.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0210] As previously described, in some embodiments, the MGS peptide selectively and / or specifically binds to lung fibroblasts and mediates internalization of the cargo to the lung fibroblasts. In various embodiments, the cargo can retain functional activity inside a lung fibroblast. For example, the MGS peptide of the MGS-cargo conjugate can bind to the outer surface of the lung fibroblast which triggers the uptake of the MGS- cargo conjugate into the lung fibroblast, and the cargo is the active component once inside the cell.

[0211] Other embodiments are directed to methods comprising administering any of the above-described MGS compounds and MGS-cargo conjugates to a subject or to a cell (e.g., a lung fibroblast) for decreasing gene expression or expressing a gene of interest. The cargo can include a nucleic acid. In some embodiments, the nucleic acid binds to RNA transcribed from the gene of interest. In some embodiments, the cargo includes a nucleic acid encoding a gene of interest associated with a protein that is expressed in response to internalization of the MGS-cargo conjugate.

[0212] Disclosed are dosing regimens comprising administering a single dose of any of the disclosed MGS peptides, MGS compounds, or MGS-cargo conjugates to a subject in need thereof, wherein the single dose comprises an amount effective to target lung fibroblasts.

[0213] Disclosed are dosing regimens comprising administering at least two doses of any of the disclosed MGS peptides, MGS compounds, or MGS-cargo conjugates to a subject in need thereof, wherein each dose is the same concentration. In some embodiments, each dose after a first dose can be decreased. In some embodiments, each dose after a first dose can be increased.

[0214] In some embodiments, a single dose can be a continuous administration. In some embodiments, a continuous administration can be hours, days, weeks, or months. In some embodiments, there can be two or more doses. In some embodiments, the two or more doses can be administered days, weeks, or months apart.

[0215] The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosedPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) are kits comprising one or more of the disclosed MGS peptides, MGS compounds, MGS-cargo conjugates, compositions, linkers, or combinations thereof. For example, disclosed are kits comprising any of the disclosed compositions.

[0216] Reference throughout the specification to “examples”, “an example”, “some examples”, and so forth, means that a particular element (e.g., feature, structure, and / or characteristic) described in connection with the example is included in the example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any embodiment may be combined in any suitable manner in the various embodiments unless the context clearly dictates otherwise.

[0217] Although specific embodiments have been illustrated and described herein, a variety of alternate and / or equivalent implementations may be substituted for the specific embodiments shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific embodiments discussed herein. Therefore, it is intended that this disclosure be limited only by the claims and the equivalents thereof.EXPERIMENTAL EMBODIMENTS

[0218] A number of experimental embodiments were directed to generating MGS peptides that selectively bind to lung fibroblasts and mediate internalization of a cargo thereto, as well as compositions (e.g., compounds and conjugates) formed therefrom. Experiments were directed to assessing the lung fibroblast specificity and internalization in vitro and in vivo.Example 1 : Dimer and Tetramer Core Formation and Cell Internalization

[0219] Below is an example of a structure of a dimer core representing MGS variants specific to normal human lung fibroblasts (NHLF) in accordance with various experimental embodiments:PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)where each X comprises an MGS peptide, and Z and Z’ comprise reactive groups. When X is SFWMILNDIYDDTPLSEFRL (SEQ ID NO: 1), the dimer structure is sometimes herein referred to as MGS NHLP V1-2. Similar structures were made with X being SEQ ID NO: 2-58, among other MGS peptide sequences as further described herein. The use of the PEG linker, in various experiments, provides solubility.

[0220] Below is an example of a structure of a tetramer core representing MGS variants in accordance with various experimental embodiments:where each X comprises an MGS peptide, and Z and Z’ comprising reactive groups.When X is SFWMILNDIYDDTPLSEFRL (SEQ ID NO: 1), the structure is sometimes herein referred to as MGS NHLP V1-4. Similar structures were made with X being SEQ ID NO: 2-58.

[0221] In various experiments, Z was either:PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) Dye (e.g., AF647), andZ’ was H. As may be appreciated and sometimes used herein, “B” stands for biotin and “AF” stands for Alex Fluor.

[0222] The tetramers were made by linear synthesis. For example, MGS NHLF Vl- 4 AF647 refers to a tetramer formed using for MGS NHLF Vl and made by linear synthesis and conjugated to AF647. The following provides further guidance on the naming convention used herein for the peptides, multimers, and conjugates. The “V#” after MGS NHLF is the peptide name (e.g., MGS NHLF Vl) and refers to the peptide sequence (e.g., VI refers to SEQ ID NO: 1). As described above, the number after “V#” in the peptide name (e.g., the “4” in Vl-4) refers to the number of peptides, thereby identifying the composition as a monomer or multimer. Any additional components, which can be used to form conjugates, are listed after “V#-#”. For example, MGS NHLF V1-1 B refers to the MGS-cargo conjugate that is specific to lung fibroblasts and includes the MGS peptide of SFWMILNDIYDDTPLSEFRL (SEQ ID NO: 1) in a monomer form and is conjugated to biotin. MGS NHLF V1-2 B refers to the MGS-cargo conjugate that is specific to lung fibroblasts and includes MGS peptide of SFWMILNDIYDDTPLSEFRL (SEQ ID NO: 1) in a dimer form and is conjugated to biotin.

[0223] All MGS peptides were made by fluorenylmethoxycarbonyl protecting group (Fmoc) solid phase peptide synthesis. PEG linkers (e.g., PEG12) were incorporated between the MGS peptide and the linker structure. Peptides were purified by reversed- phased high performance liquid chromatography (RP-HPLC), analyzed for purity using an analytical RP-HPLC, and characterized by mass spectrometry. Peptide purification was performed using an Agilent 1260 Infinity II LC system with 0.1 percent (%)Trifluoroacetic acid (TFA) in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a Kinetex XB-C18 (Phenomenex, 5 micrometer (pm), 100 angstrom (A), 250x21.2 millimeter (mm)) column at a flow rate of lOmL / min using a 30-70% B gradient over 40 minutes at roomPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) temperature. Ultraviolet (UV) absorbance at 214 nanometers (nm) was monitored. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through either a lupiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column or a Kinetex Cl 8 (Phenomenex, 5 m, 100A, 150x4.6mm) column at a flow rate of 1 milliliter (mL) / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm was monitored. Mass spectra was obtained from a 4000 QTrap MS / MS System in Q3 with a scan rate of 250 m / z / sec for >10 cycles. Machine parameters were set using a Declustering Potential (DP) between 50-100, an Entrance Potential (EP) of 10, a Collision Cell Exit Potential (CXP) of 25, a Curtain Gas (Cur) of 25, an Ion-Spray Voltage (IS) of 5000, a Temperature (TEM) at 0, the Ion Source Gas 1 at 20, the Ion Source Gas 2 at 5, and with the Interface Heater (ihe) ON.

[0224] In various experiments, the following MGS dimer was prepared:which may be referred to as MGS_NHLF_V1-2_B. The dimer was synthesized by linear Fmoc solid phase peptide chemistry and tags were added by the addition of a thiol group with maleimide biotin (or AF647 for the tetramer).

[0225] FIGs. 2A-2B show the analytical reverse-phase high-performance liquid chromatography (RP-HPLC) (FIG. 2A) and electrospray ionization Mass Spectrometry (MS) (FIG. 2B) results of the MGS NHLF V1-2 B structure.

[0226] In various experiments, the NHLF were exposed to MGS peptides to assess for lung fibroblast binding. The peptide sequence was selected by biopanning performed on NHLP. Phage library expressed 20mer peptide fused to the N-terminus of the phage pill protein.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0227] FIGs. 3 A-3D show the quantitative flow cytometry assay results for MGS peptide internalization gating. Specifically, a cell sample that was untreated was compared to a cell sample treated with MGS_NHLF_V1-2_B. FIG. 3A are graphs showing forward scatter (FSC) verses side scatter (SSC) gating of the untreated cells (left) and treated cells (right). FIG. 3B are graphs showing FSC-A (area) verses FSC-H (height) gating of the untreated cells (left) and treated cells (right). FIGs. 3C-3D are histogram graphs of the untreated cells (left) and treated cells (right) of AF647 parameter (FIG. 3C) with histogram set at less than or equal to 5% of untreated sample as positive. In FIG. 3D, the mean fluorescence intensity (MFI) was used to calculate the number of molecules / cell. The MFI of the untreated cells (left) was 2 and the MFI of the treated cells (right) was 246.

[0228] FIGs. 4A-4B show the quantitative flow cytometry assay results for MGS peptide internalization including the set of standardization beads. FIG. 4A is a graph showing the histograms of fluorescence intensity for beads loaded with different amounts of AF647 (beads 0-4) as normalized to the mode. FIG. 4B is a graph showing the linear relationship between MFI / bead verses Molecules of Equivalent Soluble Fluorochrome (MESF), AF647. The linear regression equation in FIG. 4B is used to calculate the number of molecules internalized by lung fibroblasts. Table 2 below summarizes the standard bead type, MESF, and MFI, and Table 3 summarizes the results of untreated cells and treated cells. As shown by FIGs. 4A-4B, quantity flow cytometry assays can be used to account for the number of molecules that internalized in cells.Table 2PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)Table 3

[0229] FIG. 5 is a graph showing the concentration curve and ECso of MGS NHLF V1-2 B after one hour incubation with NHLF. As shown, the ECso was 626.4 nanomolar (nM) after the one hour incubation with N=3.

[0230] FIGs. 6A-6B are fluorescent images of control incubations and FIG. 6C shows cellular internalization of MGS peptides by NHLF. FIG. 6A is an image of NHLF after no treatment, FIG. 6B is an image of NHLF after being treated with streptavidin (no MGS peptides), and FIG. 6C is an image of NHLF after being treated with MGS NHLP V1-2 B. In each of the images, cell membranes are shown in green, nucleus in blue, and MGS-Streptavidin-AF647 in red. For example, the cell samples in various experiments were stained with DAPI nuclear stain (shown in blue) and Wheat Germ Agglutinin (WGA)-488, which binds to membranes of cells (shown in green). The NHLF were exposed to 100 nM MGS NHLP V1-2 B and with a 1 : 1 MGS to streptavidin ratio for one hour incubation time. As shown by FIG. 6B, no internalization or cell binding is observed for streptavidin alone. FIG. 6C shows that the MGS peptide mediated the internalization.

[0231] In various experiments, the following MGS tetramer was prepared:PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)The above structure may be referred to as MGS NHLF V1-4_AF647. The tetramericMGS NHLF V1-4 was synthesized by linear Fmoc solid phase peptide chemistry, purified by RP-HPLC, and AF647 was added by the addition of a thiol group with maleimide.

[0232] FIGs. 7A-7B show the analytical RP-HPLC (FIG. 7A) and electrospray ionization MS (FIG. 7B) results of the MGS NHLF V1-4 AF647 structure.

[0233] As with the dimer, the normal human lung fibroblasts were exposed to the tetramer of the MGS peptide to assess for NHLP internalization.

[0234] FIG. 8 is a graph showing MGS NHLF V1-4 AF647 uptake by NHLF. Also shown in the graph, is the dimer (MGS NHLF V1-2 AF647) uptake by NHLF in triangles for comparison. As shown by FIG. 8, the uptake of MGS NHLF V1-4 AF647 was extremely high, reaching intracellular concentrations of greater than 1 pM. The tetramer exhibited higher uptake or internalization than the dimer version ofMGS NHLF V1. The data of FIG. 9 shows that there may be rapid receptor recycling occurring. Rapid receptor recycling confounds the ECso and Bmax measurements in the assay (e.g., dynamic system is not at equilibrium). In such cases, the absolute uptake is a better measure of performance. Without being bound by theory, it is believed the receptor is recycled by dumping the cargo in the cell and then being re-internalized.

[0235] FIG. 9 is a graph showing the internalization of MGS_NHLF_V1-4_AF647 by NHLF. This data illustrates the effect of a receptor recycling inhibitor versus a protein synthesis inhibitor on time-dependent uptake of MGS peptide. In the experiment, NHLF were exposed to MGS_NHLF_V1-4_AF647, MGS_NHLF_V1-4_AF647 +PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) cycloheximide (CHX), and MGS_NHLF_V1-4_AF647 + 250 pM hydroxychloroquine (HCQ), each at 50 nM of MGS NHLF V1-4 AF647. The rate of MGS NHLF Vl- 4_AF647 uptake by NHLF, in absence of inhibitors (e.g., CHX or HCQ), was sustained for at least four hours. No inhibition was observed by CHX, which indicates that new protein synthesis is not required to maintain the MGS peptide uptake over time. The 45% inhibition of MGS peptide uptake in the presence of HCQ suggests that the peptide receptor is normally recycled in a process that uses acidification to separate the internalized ligand from the receptor. Table 4 below provides a summary of the results.Table 4Example 2: Internalized Peptide Escape from Vesicles to Cytoplasm

[0236] Various experiments were conducted to assess whether internalized MGS_NHLF_V1-4_AF647 peptides escaped from vesicles to cytoplasm. To assess for peptide escape, NHLF were incubated with MGS_NHLF_V1-4_AF647 for various periods of time. For example, the NHLF were incubated for: (a) one hour and harvested; (b) two hours, the peptides were removed and then the isolated cells were incubated for an additional twenty -two hours and harvested; and (c) twenty -four hours continuous incubation with peptides and harvested. After the incubation, the cells were washed with phosphate-buffered saline (PBS) and acid wash solutions. The cells were harvested in PBS+10 millimolar (mM) ethylenediaminetetraacetic acid (EDTA). Each NHLF sample was divided into two, the cells were pelleted and supernatant was removed. The two divided samples of cells were then resuspended in 200 microliters (pL) of: (i) one divided sample in PBS; and (ii) the other divided sample in PBS + 0.05% saponin. The divided samples were then incubated on ice for ten minutes and 500 pL PBS + 2% Bovine serum albumin (BSA) was added and then the peptide-AF647 in cells were assayed by flow cytometry. As may be appreciated, saponin is a selective detergent, which can permeabilize the plasma membrane of cells and allow for the MGS peptide toPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) be released therefrom. Intracellular vesicle membranes are not permeabilized by saponin. MGS present in intracellular vesicles, not in cytoplasm, is retained by the cell, even in the presence of the saponin.

[0237] FIGs. 10 A- 10C show the flow cytometry results indicating internalized MGS_NHLF_V1-4_AF647 peptides escape from vesicles to cytoplasm. FIG. 10A is a graph showing the flow cytometry results of cells incubated with the MGS peptide for one hour and harvested. FIG. 1 OB is a graph showing the flow cytometry results of cells incubated with the MGS peptide for two hours, the peptides were removed and then the isolated cells were included for an additional twenty -two hours and harvested. FIG. 10C is a graph showing the flow cytometry results of cells incubated with the MGS peptide for twenty-four hours and harvested. The standard flow conditions are shown in pink and the saponin treatment (permeabilization) is shown in blue. A shift to the left for the saponin treatment as compared to the standard flow conditions shows loss of MGS peptides from the cytoplasm. Table 5 below provides a summary of the results.Table 5

[0238] FIGs. 11 A-l IB are microscopy images of NHLF samples after incubation with AF647 (control, FIG. 11 A) and MGS_NHLF_V1-4_AF647 (FIG. 11B). FIG. 11B shows internalization of MGS_NHLF_V1-4_AF647 (red) by the NHLF.

[0239] FIGs. 12A-12D are four versions of the same microscopy image and associated histograms showing the MGS_NHLF_V1-4_AF647 peptides in vesicles to highlight the intensity distribution of pixels where AF647 is detected in the cell. High fluorescent pixel intensity indicates that the internalized peptides is in vesicles, whereas low fluorescent pixel intensity indicates that the peptide is distributed in the cytoplasm of thePCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) cell. FIG. 12A is a microscopy image and histogram graph of a raw image of the MGS NHLF V1-4 AF647 after incubation with NHLF. FIG. 12B is a scaled microscopy image and histogram graph of the MGS NHLF V1-4 AF647 after incubation with NHLF. The punctate pattern demonstrates that the brightest pixels for the AF647 channel are associated with intracellular vesicles. FIG. 12C and FIG. 12D are microscope images and histogram graphs of the MGS NHLF V1-4 AF647 after incubation with NHLF and after adjusting to show pixels above a threshold associated with untreated cells (no AF647), including a threshold of 1986 (FIG. 12C) and greater than 1986 (FIG. 12D). MGS peptides in the images are shown in red. In FIGs. 12C and 12D, the colors indicating cell membranes (previously shown in green) and nucleus (previously shown in blue) have been turned off. The positive pixels in FIG. 12D have a fluorescent intensity above the threshold value. The enhancement to the pixels in FIGs. 12C-12D was used to illustrate the difference between bright vesicles and the less intense, yet above threshold, pixels indicating cytoplasmic distribution of some MGS peptide NHLF.

[0240] FIGs. 13A-13C are microscopy images of NHLF alone (FIG. 13A), NHLF incubated with AF647 (FIG. 13B), and NHLs incubated with MGS NHLF Vl- 4 AF647 (FIG. 13C), which were imaged after one hour of incubation. Only pixels with fluorescence intensity above the threshold, e.g., 1986, of untreated cells are shown in red. Cells incubated with MGS_NHLF_V1-4_AF647 showed positive pixels in the pattern of intracellular vesicles.

[0241] FIGs. 14A-14C are the same microscopy images from FIGs. 13A-13C of NHLF alone (FIG. 1 A), normal NHLF incubated with AF647 (FIG. 14B), and NHLF incubated with MGS_NHLF_V1-4_AF647 (FIG. 14C) but with a change in setting of the intensity threshold to enable visualization of modest intensity pixels (e.g., above background) that are outside of intracellular vesicles. The NHLF cells were incubated for one hour with the MGS peptide and then the MGS peptide was removed, NHLF cells were washed, and then the NHLF cells were incubated for an additional twenty- three hours before being imaged. Only pixels with fluorescence intensity above the threshold (e.g., 1986) of untreated cells are shown in red. Cells incubated withPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)MGS NHLF V1-4 AF647 showed positive pixels dispersed throughout the cell (cytosolic). The brightest pixels still showed the pattern of intracellular vesicles.

[0242] FIGs. 15A-15D are microscopy images of NHLF after incubation with MGS_NHLF_V1-4_AF647 and which are adjusted to show pixels above different thresholds. FIG. 15A is an image showing pixels above 1986, FIG. 15B is an image showing pixels above 3972, FIG. 15C is an image showing pixels above 7944, and FIG. 15D is an image showing pixels above 14999. Only pixels with fluorescence intensity above the respective threshold are illustrated in each image in red, with the threshold increasing from FIG. 15A to FIG. 15D. Cells incubated with AF647 showed positive pixels dispersed throughout the cell (cytosolic). The brightest pixels still showed the pattern of intracellular vesicles.Example 3: Cell of Interest Uptake of MGS Peptides and Specificity Assessment

[0243] Various experiments were directed to assessing different cells of interest for MGS peptide uptake and specificity. The cells of interest included: (i) NHLF as primary NHLF, which were obtained from Lonza™, catalog number CC-2512; (ii) mouse pulmonary fibroblasts (MPF) as primary mouse lung fibroblast, which were obtained from Sciencell™, catalog number M3300-57; and (iii) LL29 as human idiopathic pulmonary fibrosis (IPF) lung fibroblasts, ATCC CCL-134. Internalization of MGS_NHLF_V1-4_AF647 by NHLF was illustrated previously by the graph of FIG. 8.

[0244] FIG. 16 is a graph showing internalization of MGS_NHLF_V1-4_AF647 by LL29 cells. As shown, the MGS_NHLF_V1-4_AF647 exhibited high uptake by LL29 cells.

[0245] FIG. 17 is a graph showing internalization of MGS_NHLF_V1-4_AF647 by MPF cells. As shown, the MGS_NHLF_V1-4_AF647 exhibited high uptake by MPF cells.

[0246] Based on the assessment, tetramerization of the MGS NHLF Vl improved the uptake by NHLF as compared the dimeric form, showing a greater than two-fold improvement, which is suggestive of multivalent binding on the cell surface. The peptide was internalized at a high level in lung fibroblasts from a human (500,000 / cell in one hour) and mouse (200,000 / cell), as well as lung fibroblasts isolated from a patientPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) with idiopathic pulmonary fibrosis ( 118,000 / cell in LL29 cells), as shown by FIGs. 8, 16, and 17. The MGS NHLF Vl peptide had lower uptake in human hepatocytes (5,000 / cell, 87-fold lower) and renal epithelial cells (23,000 / cell, 19-fold lower). The data is consistent with rapid recycling of the cellular receptor for the MGS peptide, resulting in high intracellular concentrations. Rapid recycling confounds ECso and Bmax measurements in this assay (dynamic system not at equilibrium), and absolute uptake is a better measure of performance. The MGS peptide escaped from vesicles into the cytoplasm over time as demonstrated by at least FIGs. 10A-15D.Example 4: Variants of MGS NHLF Vl

[0247] Variants of MGS NHLF Vl were generated by N-terminal blocking and alanine substitutions, e.g., adding an acetyl group on the N-terminus and / or replacing amino acids at different amino acids with alanine. The generated variants were assessed for solubility in aqueous solutions, binding to lung fibroblasts, and internalization by the lung fibroblasts. FIGs. 18A-18F and Tables 6-12 summarize the results of the variant assessment. In such embodiments, an acetyl group was added on the N-terminus and different amino acids were substituted with alanine to identify which amino acid positions were essential and nonessential for binding to NHLF and for solubility.Table 6: Alanine group variantsPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191.l l l (SRI-230091)Table 7: N-terminal blocking and alanine group substitution variants uptake by NHLFPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191.l l l (SRI-230091)Table 8: Individual alanine substitutions variants from 20.9 AS2 uptake by NHLFTable 9: Individual alanine substitutions variants from 20.9 AS3 uptake by NHLFPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191.l l l (SRI-230091)Table 10: Individual alanine substitutions variants from 20.9 AS4 uptake by NHLFPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191.l l l (SRI-230091)Table 12: Individual alanine substitutions variants from 20.9 AS1 uptake by NHLFPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0248] FIGs. 18A-18F are graphs showing MGS peptides uptake by NHLF. For each experiment, NHLF were incubated with the respective MGS peptide at a 50 nM concentration for one hour, with N= 3. FIG. 18A is a graph showing the uptake of the MGS peptides by NHLF in Table 7. Variants 20.9 AS2 and AS4 were insoluble. FIG. 18B is a graph showing the uptake of the MGS peptides by NHLF in Table 8. Variants 20.9 AS2 and AS8 were insoluble. FIG. 18C is a graph showing the uptake of the MGS peptides by NHLF in Table 9. Variants 20.9 AS3 and AS15 were insoluble. FIG. 18D is a graph showing the uptake of the MGS peptides by NHLF in Table 10. Variants 20.9 AS4, AS 10, and ASH were insoluble. FIG. 18E is a graph showing the uptake of the MGS peptides by NHLF in Table 11. Variants 20.9 AS19 and AS21 were insoluble. FIG. 18F is a graph showing the uptake of the MGS peptides by NHLF in Table 12, along with MGS peptides MGS_NHLF_V28 (truncated), MGS_NHLF_V29, and MGS NHLF V30 from Table 13. Table 13 below summarizes all MGS peptides.Table 13PCT / US24 / 5564213 November 2024 (13.11.2024)S1647.191.lll (SRI-230091)PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)*the multimer core of MGS NHLF V30 included an additional reactive group of sstBU. The reactive groups (e.g., cys and / or sstBU) allow for attachment to cargo, such as attaching two cargos on MGS NHLF V30 (e.g., dye and a therapeutic or other cargo).

[0249] The above-described alanine substitutions and N-blocking was used to produce and assess twenty-three distinct variants of the NHLF-specific MGS tetrameric peptide and to assess for solubility and uptake by NHLF relative to the parental tetramer. N- terminal acetylation of the peptide, e.g., MGS_ NHLF V2-4, did not alter peptide binding and internalization. Group alanine substitutions showed that residues toward the C-terminus were not required for binding and uptake. Single alanine substitutions showed that six residues for important for peptide solubility. Further, single alanine substitutions for Ile-9 showed this residue is required for binding and internalization, for Tyr-10 showed this residue is important for binding and internalization, and for Leu-7 showed this residue has a negative effect on binding and internalization.

[0250] Based on the above, an MGS peptide with the sequence of CH3CO- SFWAIANDIYD (SEQ ID NO: 56) was assessed. This MGS peptide was based on MGS_ NHLF _V2 (SEQ ID NO: 2) which included:CH3CO-SFWMILNDIYDDTPLSEFRLIn the above experiments and more specifically, acetylation was determined to not impact uptake by NHLF. Changes to amino acids of Ser-1, Trp-3, Asn-7, Asp-8, Asp- 11, and Pro- 14 were determined to impact solubility (e.g., change to alanine resulted in insolubility). Amino acids of Ser-1 and Trp-3 were likely required for binding based on the alanine block substitution. Amino acids Ile-9 and Tyr-10 were determined as being essential by single alanine substitutions. Amino acids Phe-2, Met-4, Ile-5, and Leu-6 were determine as having a negative contribution to binding, with L-6 have the highestPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) negative impact to binding. Amino acids of Asp-12, Thr-3, Leu-15, Ser-16, Glu-17, Phe-18, Arg- 19, and Leu-20 were determined as being nonessential based on alanine block substitutions and single alanine substitutions. As may be appreciated and as used throughout, the three letters (e.g., “Ser-”) refers to the amino acid and therefers to the position of the amino acid in the sequence. For example, Ser-1 refers to the amino acid of serine which is in position 1 of the sequence.

[0251] In the MGS peptide of CH3CO-SFWAIANDIYD (SEQ ID NO: 56), Met-4 and Leu-6 were changed to Ala and the C-terminus of DTPLSEFRL was truncated. If the C- terminus truncation is insoluble, the C-terminus could be added back. Phe-2 was change to Ala only if necessary.Example 5: Serum Stability Assessment

[0252] In various experiments, the different MGS peptides in tetramer form were assessed for stability in human serum and mouse serum.

[0253] FIG. 19 shows the analytical RP-HPLC (left and middle) and electrospray ionization MS (right) results of the MGS_NHLF_V1-4_AF647 structure after incubation in 50% human serum for various times. Results indicate the MGS peptide is stable in human serum. The MGS_NHLF_V1-4_AF647 was placed in human serum at a concentration of 60pM at 37 degrees Celsius (C) and assessed at indicated time points over forty-eight hours. At the indicated time points (e.g., 0 hour, 1 hour, 2 hour, 4 hour, 6 hour, 24 hour, and 48 hour), a portion of the sample was removed and serum proteins were precipitated in ethanol. The stability of the peptide was monitored using RP-HPLC with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each time point injection was run through either a lupiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate of ImL / min using a 20- 70%B gradient over 20 min at room temperature. UV absorbance at 214nm and 651nm were monitored. A mass was observed at 13721.78 Daltons (Da). Even with the free amino terminus, the MGS_NHLF_V1-4_AF647 peptide was stable in human serum at 37 degrees C.

[0254] FIGs. 20A-20D show details of human serum stability assessment results of MGS NHLF V29-4 AF647. The MGS_NHLF_V29-4_AF647 was placed in 50%PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) human serum / PBS at a concentration of 60pM at 37 degrees Celsius (C) and assessed at time points of 0 hour, 1 hour, 6 hours, and 24 hours. As described above, at the indicated time points, a portion of the sample was removed, and serum proteins were precipitated in ethanol. The stability of the dimer MGS_NHLF_V29-4_AF647 was monitored on RP-HPLC (as described above) under UV absorbance at 651nm. FIG. 20A shows the structure of MGS_NHLF_V29-4_AF647. FIG. 20B shows the electrospray ionization MS results of the MGS_NHLF_V29-4_AF647 structure. A mass was observed at 13475.625 Da (0.025% error). FIG. 20C is a graph showing the intact peptide % over time after being placed in the human serum / PBS. FIG. 20D shows the analytical RP-HPLC results, with the stars indicating material is remaining.

[0255] FIGs. 21 A-21D show details of mouse serum stability assessment results of MGS NHLF V2-4 AF647. The MGS_NHLF_V2-4_AF647 was placed in 50% mouse serum / PBS at a concentration of 60pM at 37 degrees C and assessed at time points of 0 hour, 1 hour, 6 hours, and 24 hours. As described above, at the indicated time points, a portion of the sample was removed and serum proteins were precipitated in ethanol. The stability of the dimer MGS_NHLF_V2-4_AF647 was monitored on RP-HPLC (as described above) under UV absorbance at 651nm. FIG. 21 A shows the structure of MGS NHLF V2-4 AF647. FIG. 2 IB shows the electrospray ionization MS results of the MGS_NHLF_V2-4_AF647 structure. A mass was observed at 13886.62 Da (0.0089% error). FIG. 21C is a graph showing the intact peptide % over time after being placed in the mouse serum / PBS. FIG. 21D shows the analytical RP-HPLC results, with the stars indicating material is remaining.

[0256] FIG. 22A-22C show details of mouse serum stability assessment results of MGS NHLF V29-4 AF647. The MGS_NHLF_V29-4_AF647 was placed in 50% mouse serum / PBS at a concentration of 60pM at 37 degrees C and assessed at time points of 0 hour, 1 hour, 6 hours, and 24 hours. As described above, at the indicated time points, a portion of the sample was removed and serum proteins were precipitated in ethanol. The stability of the dimer MGS_NHLF_V29-4_AF647 was monitored on RP-HPLC (as described above) under UV absorbance at 651nm. FIG. 22A shows the electrospray ionization MS results of the MGS_NHLF_V2-4_AF647 structure. A mass was observed at 13478.69 Da (0.0029% error). FIG. 2 IB is a graph showing the intactPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) peptide % over time after being placed in the mouse serum / PBS. FIG. 21C shows the analytical RP-HPLC results, with the stars indicating material is remaining.

[0257] FIGs. 23A-23E show example fragments of MGS NHLF V2-4 AF647 that arose during incubation of MGS_NHLF_V2-4_AF647 with mouse serum. The fragments included CH3O-SFWMILN-DIYDDTPLSEFRL (referred to as “Z” and exhibiting an observed mass of 13067 Da (-820 Da), CH3O- SFWMILNDIYDDTPLSEFR-L (referred to as “X” and exhibiting an observed mass of 11500 Da (-2388 Da), and CH3O-SFWMILNDIYDDTPLSEF-RL (referred to as “A” and exhibiting an observed mass of 11656 Da (-2231 Da). FIGs. 23 A-23C show the structure of fragment Z (FIG. 23 A), fragment X (FIG. 23B), and fragment A (FIG. 23C). FIG. 23D shows the electrospray ionization MS results of the fragments. FIG. 23E shows the analytical RP-HPLC results, with the circles showing the peptide fragments.

[0258] FIGs. 24A-24E show example fragments of MGS_NHLF_V29-4_AF647 that arose during incubation of MGS_NHLF_V29-4_AF647 with mouse serum. The fragments included CH3O-SFWAIA-NDIYDDTPLSEFRL (referred to as “Z” and exhibiting an observed mass of 12758 Da (-720 Da), CH3O- SFWAIANDIYDDTPLSEFR-L (referred to as “X” and exhibiting an observed mass of 11193 Da (-2285 Da), and CH3O-SFWAIANDIYDDTPLSEF-RL (referred to as “A” and exhibiting an observed mass of 11349 Da (-21291 Da). FIGs. 24A-24C show the structure of fragment Z (FIG. 24A), fragment X (FIG. 24B), and fragment A (FIG. 24C). FIG. 24D shows the electrospray ionization MS results of the fragments. FIG. 24E shows the analytical RP-HPLC results, with the circles showing the peptide fragments.

[0259] As shown by the above-described results, the parental MGS NHLF V2- 4_AF647 and MGS_NHLF_V29-4_AF647 had similar stability half-life and degradation patterns as assessed by HPLC. The peptide cleavage is likely occurring at the same location in both peptides and is not altered by the substitutions of alanine in positions 4 and 6. The MGS_NHLF_V29-4_AF647 half-life is approximately 10-fold greater in human serum than mouse serum, which is likely due to mouse serum being more proteolytic than human serum. The HPLC chromatograms suggests that an internalPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) cleavage site and not peptidase degradations from the N- or C-terminus. The MS and MS / MS sequencing is consistent with loss of a peptide branch either at Phe-18 or Arg- 19. Based on this, it is believed that the MGS is likely to maintain activity as a trimer or dimer such that even after loss of a peptide branch, the MGS should maintain ability to target NHLF. As the cleavage occurs at a discrete location, the peptide can be optimized at this region to improve serum stability.Example 6: In vivo delivery to tissue

[0260] Various experiments were conducted to assess for in vivo delivery of the MGS peptides to tissue. In such experiments, an IVIS instrument was used for collecting a near infrared (NIR) signal from the full thickness of mouse organs, including the lungs, heart, and spleen. The IVIS signal is not from the intracellular MGS peptide. In the various experiments, C57BL / 6 mice were injected intravenously (i.e., IV) through the tail vein with 1 micrograms (pg) / g animal weight of the MGS peptide in 100 microliters (pL) PBS. Twenty-four hours post injection, the mice were imaged via ex vivo NIR imaging.

[0261] FIG. 25 shows images of heart, lung, and spleen, from an untreated mouse (control) and mice treated with MGS_NHLF_V2-4_AF647. In each image, a region of interest (ROI) is drawn around organs to determine the total radiant efficiency (e.g., level of fluorescence) for each organ. More specifically, FIG. 25 includes images of organs from the control mouse that was not injected with an MGS peptide and 3 sets of images of organs obtained from mice injected with MGS_NHLF_V2-4_AF647. The three mice injected with MGS_NHLF_V2-4_AF647 had higher ROIs for the lungs compared to the control mouse, while the ROIs for the heart and spleen were similar between the MGS-treated mice and the control mouse.

[0262] FIG. 26 shows images of heart, lung, and spleen from an untreated mouse (control) and mice treated with MGS_NHLF_V29-4_AF647. In each image, a ROI is drawn around organs to determine the total radiant efficiency (e.g., level of fluorescence) for each organ. More specifically, FIG. 26 includes images of organs from the control mouse that was not injected with an MGS peptide and 3 sets of images of organs obtained from mice injected with MGS_NHLF_V29-4_AF647. The three micePCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) injected with MGS NHLF V29-4 AF647 had higher ROIs for the lungs compared to the control mouse, while the ROIs for the heart and spleen were similar between the MGS-treated mice and the control mouse.

[0263] FIG. 27 is a summary graph showing the ex vivo MGS peptide distribution in isolated organs as imaged by IVIS NIR.Example 7: In vivo uptake- fibroblast isolation

[0264] A single cell suspension from mouse lungs was prepared as follows. After image collection of the intact lung (e.g., FIGs. 25-26), tissue was minced using a handheld razor blade. The minced tissue was transferred to a Miltenyi “gentleMACS C” tube with 8 ml Dulbecco’s Modified Eagle Medium (DMEM) containing 1 milligram (mg) / mL collagenase I and 1 mg / mL collagenase II. The dissociator tube was capped and loaded into the tissue dissociator with the heating cover to maintain 37 degrees C. The digestion cycle was run for thirty minutes with continuous mixing.

[0265] At the end of the incubation, the sample was diluted to 40 mL with PBS containing 2% fetal bovine serum (FBS) and undigested tissue chunks were removed by passing the sample though a 100 micrometer (pm) filter. Cells were pelleted at 1000 x g for five minutes and supernatant removed and discarded. Cell pellet was treated with Ammonium-Chloride-Potassium (ACK) lysing buffer if red blood cells were evident. Cells were washed in PBS and repelleted. The pellet was resuspended in 1 mL PBS + 10 mM EDTA and run on the flow cytometer. Such protocols were implemented as described in Green et al. and Matsushima et al. See Green et al., Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor a Kinase Activity, Am I Respir Cell Mol Biol, 54:4, pp 534-545 (April 2016) ; Matsushima et al., CD248 and Integrin alpha-8 are candidate markers for differentiating lung fibroblast subtypes, BMC Pulmonary Medicine, 20:21, (2020), each of which are hereby incorporated by reference in their entirety for their teachings.

[0266] In some prior work (e.g., Green), the lung fibroblasts were separated (Linneg) from the bulk of the cell population based on absence of staining for other lineage markers. See at least FIG. 1 A of Green. The markers included: CD45posimmune cells, CD3 lposendothelial cells, and CD326posepithelial cells. In other prior work (e.g.,PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)Matsushima), the lung fibroblasts were also separated (Linneg) from the bulk of the cell population based on absence of staining for other lineage markers. See at least FIG. 1 A of Matsushima. The markers included: CD31posvascular endothelial cells, CD45poshematopoietic cells, CD146pospericytes and smooth muscle cells, E-cadherinpos(epithelial cells), LYVElneg(lymphatic endothelial cells), and TER-119+(erythrocytes). The Linnegcells were further divided by intensity of PDGFRA staining into negative, low, and high subsets as well as Sca-1 low and high staining groups.Example 8: In vivo delivery to lung fibroblasts

[0267] Various embodiments were directed to the assessment of in vivo delivery of the MGS peptides to lung fibroblasts. Flow cytometry is useful for estimating the level of peptide internalization by individual cells and identifying the cells of interest in a pool of multiple cell types. The number of molecules / cell is likely an underestimate of the true number that can be internalized due to peptide clearance from the blood and the breakdown of peptide in cells and release of AF647 from the cells.

[0268] FIGs. 28A-28F show the results of flow cytometry gating of single cells which were isolated from mouse lung tissue. As noted above, the mouse tissue was collected twenty-four hours after peptide injection, as described above in connection with Examples 6-7. FIG. 28A is a graph of the flow cytometry results including FSC-A verses SSC-A. FIGs. 28B-28C are graphs showing R-phychoerythrin (PE) flow cytometry results for an isotype control (FIG. 28B) for the lineage-specific cell surface markers of CD 16 / 32, CD31, CD45, and CD326 (FIG. 28C). FIGs. 28C shows the cells of interest (e.g., PEneg). FIGs. 28D-28E are graphs showing fluorescein isothiocyanate (FITC) flow cytometry results for an isotype control (FIG. 28D) and for the lineagespecific cell surface markers CD140a (FIG. 28E). FIG. 28E shows the cells of interest (e.g., FITCpos). FIG. 28F is a graph showing the cells of interest, e.g., the PEnegFITCposcells that include lung fibroblasts. The cells of interest were approximately 0.3% to 1% of the single cell preparation.

[0269] FIGs. 29A-29B show the results of flow cytometry from lung fibroblasts (PEneg / FITCp°s cells) isolated from mouse lung tissue collected twenty-four hours after injection with MGS_NHLF_V2-4_AF647 (FIG. 29 A) and MGS_NHLF_V29-4_AF647PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)(FIG. 29B). As described above, the lung fibroblasts were cells that were PEnegFITCpos.Table 14 below summarizes the results:Table 14

[0270] FIGs. 30A-30B show the results of flow cytometry from unknown cells (PEneg / FITCneg) isolated from mouse lung tissue collected twenty-four hours after injection with MGS_NHLF_V2-4_AF647 (FIG. 30 A) and MGS_NHLF_V29-4_AF647 (FIG. 30B). The unknown cells were PEnegFITCneg. As shown by FIGs. 29A-30B, the MGS peptides were internalized greater by the lung fibroblasts as compared to the unknown cells. Table 15 below summarizes the results:Table 15

[0271] Overall, the MGS_NHLF_V2-4_AF647 and MGS_NHLF_V29-4_AF647 showed similar stability half-life (3-4 hours) in mouse serum. The MGS peptides exhibited tissue specific delivery to the lungs and were more efficiently delivered by IVPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) injections as compared to SubQ route of administration. In isolated lung fibroblasts, the highest level of AF647 retention was observed with MGS_NHLF_V29-4 AF647. The results show that the MGS peptides can be useful in carrying therapeutic cargo in the blood to be taken in by lung fibroblasts.Example 9: Synthesis Scheme

[0272] Various experiments were directed to synthesizing different MGS peptides, including those described above in Table 1.

[0273] FIGs. 31A-31C show an example structure of MGS_NHLF_V8-4_AF647 and analytical results of the structure. More specifically, FIG. 31A shows the MGS_NHLF_V8-4_AF647, which included a tetramer of the MGS peptide of SFWMILNDIYDDTPLAEFRL conjugated to AF647. FIG. 3 IB is the resulting analytical RP-HPLC and FIG. 31C is the electrospray ionization MS results of the MGS_NHLF_V8-4_AF647. The calculated mass was 13655.71 Da, solubility of n / a in H2O and greater than 13 pM in 5 mM ammonium bicarbonate (AB), and yield of around 1 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a lupiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0274] FIGs. 32A-32C show an example structure of MGS_NHLF_V9-4_AF647 and analytical results of the structure. More specifically, FIG. 32A shows the MGS_NHLF_V9-4_AF647, which included a tetramer of the MGS peptide of SFWMILNDIYDDTPASEFRL conjugated to AF647. FIG. 32B is the resulting analytical RP-HPLC and FIG. 32C is the electrospray ionization MS results of the MGS_NHLF_V9-4_AF647. The calculated mass was 13551.38 Da, solubility of n / a in H2O and greater than 17 pM in 10 mM AB, and yield of around 1 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a upiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow ratePCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191.l l l (SRI-230091) of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0275] FIG. 33 shows an example structure of MGS_NHLF_V10-4_AF647. More specifically, FIG. 33 shows the MGS_NHLF_V10-4_AF647, which included a tetramer of the MGS peptide of SFWMILNDIYDDTALSEFRL conjugated to AF647. The calculated mass was 13615.55 Da. The MGS_NHLF_V10-4_AF647 crude peptide did not dissolve in 60% Acetonitrile.

[0276] FIGs. 34A-34C show an example structure of MGS NHLF Vl 1-4 AF647 and analytical results of the structure. More specifically, FIG. 34A shows the MGS NHLF Vl 1-4 AF647, which included a tetramer of the MGS peptide of SFWMILNDIYDDAPLSEFRL conjugated to AF647. FIG. 34B is the resulting analytical RP-HPLC and FIG. 34C is the electrospray ionization MS results of the MGS NHLF Vl 1-4 AF647. The calculated mass was 13599.6 Da, solubility of n / a in Marks Quality H2O (MQH2O) and greater than 20 pM in 10 mM AB, and yield of 2.5 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a lupiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0277] FIG. 35 shows an example structure of MGS_NHLF_V12-4_AF647. More specifically, FIG. 35 shows the MGS_NHLF_V12-4_AF647, which included a tetramer of the MGS peptide of SFWMILNAIYDDTPLSEFRL conjugated to AF647. The calculated mass was 13543.67 Da and yield of 2 mg. The MGS_NHLF_V12-4_AF647 did not dissolve in 6M GuHCL.

[0278] FIG. 36 shows an example structure of MGS_NHLF_V13-4_AF647. More specifically, FIG. 36 shows the MGS_NHLF_V13-4_AF647, which included a tetramer of the MGS peptide of SFWMILADIYDDTPLSEFRL conjugated to AF647. The calculated mass was 13547.6 Da. The MGS_NHLF_V14-4_AF647 crude peptide did not dissolve in 60% Acetonitrile.

[0279] FIGs. 37A-37C show an example structure of MGS_NHLF_V14-4_AF647 and analytical results of the structure. More specifically, FIG. 37A shows thePCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)MGS_NHLF_V14-4_AF647, which included a tetramer of the MGS peptide of SFWMIANDIYDDTPLSEFRL conjugated to AF647. FIG. 37B is the resulting analytical RP-HPLC and FIG. 37C is the electrospray ionization MS results of the MGS_NHLF_V14-4_AF647. The calculated mass was 13551.38 Da, solubility of 1.2 pM in MQH2O and greater than 30 pM in 5 mM AB, and yield of around 1 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a lupiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0280] FIGs. 38A-38C show an example structure of MGS NHLF V15-4 AF647 and analytical results of the structure. More specifically, FIG. 38A shows the MGS NHLF V15-4 AF647, which included a tetramer of the MGS peptide of SFWMALNDIYDDTPLSEFRL conjugated to AF647. FIG. 38B is the resulting analytical RP-HPLC and FIG. 38C is the electrospray ionization MS results of the MGS_NHLF_V15-4_AF647. The calculated mass was 13551.38 Da, solubility of n / a in MQH2O and 24 pM in 5 mM AB, and yield of around 1 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a upiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0281] FIGs. 39A-39C show an example structure of MGS_NHLF_V16-4_AF647 and analytical results of the structure. More specifically, FIG. 36A shows the MGS NHLF V16-4 AF647, which included a tetramer of the MGS peptide of SFWMILNDIYDATPLSEFRL conjugated to AF647. FIG. 36B is the resulting analytical RP-HPLC and FIG. 36C is the electrospray ionization MS results of the MGS_NHLF_V16-4_AF647. The calculated mass was 13543.67 Da, solubility of n / a in MQH2O and greater than 12 pM in 10 mM AB, and yield of around 0.5 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection wasPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091) run through a Jupiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0282] FIG. 40 shows an example structure of MGS_NHLF_V17-4_AF647. More specifically, FIG. 40 shows the MGS_NHLF_V17-4_AF647, which included a tetramer of the MGS peptide of SFWMILNDIYADTPLSEFRL conjugated to AF647. The calculated mass was 12558.67 Da. The MGS_NHLF_V17-4_AF647 did not dissolve in 6M GuHCL.

[0283] FIGs. 41A-41C show an example structure of MGS_NHLF_V18-4_AF647 and analytical results of the structure. More specifically, FIG. 41 A shows the MGS_NHLF_V18-4_AF647, which included a tetramer of the MGS peptide of SFWMILNDIADDTPLSEFRL conjugated to AF647. FIG. 4 IB is the resulting analytical RP-HPLC and FIG. 41C is the electrospray ionization MS results of the MGS_NHLF_V18-4_AF647. The calculated mass was 12366.32 Da, solubility of n / a in MQH2O and greater than 35 pM in 10 mM AB, and yield of 1 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a Jupiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0284] FIGs. 42A-42C show an example structure of MGS_NHLF_V19-4_AF647 and analytical results of the structure. More specifically, FIG. 42A shows the MGS_NHLF_V19-4_AF647, which included a tetramer of the MGS peptide of SFWMILNDAYDDTPLSEFRL conjugated to AF647. FIG. 42B is the resulting analytical RP-HPLC and FIG. 42C is the electrospray ionization MS results of the MGS_NHLF_V19-4_AF647. The calculated mass was 13551.38 Da, solubility of n / a in MQH2O and 37 pM in 10 mM AB, and yield of around 1 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a Jupiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate ofPCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0285] FIGs. 43A-43C show an example structure of MGS_NHLF_V20-4_AF647 and analytical results of the structure. More specifically, FIG. 43 A shows the MGS_NHLF_V20-4_AF647, which included a tetramer of the MGS peptide of SFWAILNDIYDDTPLSEFRL conjugated to AF647. FIG. 43B is the resulting analytical RP-HPLC and FIG. 43C is the electrospray ionization MS results of the MGS_NHLF_V20-4_AF647. The calculated mass was 13479.25 Da, solubility of n / a in MQH2O and greater than 35 pM in 10 mM AB, and yield of around 1 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a lupiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0286] FIG. 44 shows an example structure of MGS_NHLF_V21-4_AF647. More specifically, FIG. 44 shows the MGS_NHLF_V21-4_AF647, which included a tetramer of the MGS peptide of SFAMILNDIYDDTPLSEFRL conjugated to AF647. The calculated mass was 12274.16 Da. The MGS_NHLF_V21-4_AF647 crude peptide did not dissolve in 60% Acetonitrile.

[0287] FIGs. 45A-45C show an example structure of MGS_NHLF_V22-4_AF647 and analytical results of the structure. More specifically, FIG. 43 A shows the MGS_NHLF_V22-4_AF647, which included a tetramer of the MGS peptide of SAWMILNDIYDDTPLSEFRL conjugated to AF647. FIG. 45B is the resulting analytical RP-HPLC and FIG. 45C is the electrospray ionization MS results of the MGS_NHLF_V22-4_AF647. The calculated mass was 12430.31 Da, solubility of n / a in MQH2O and greater than 25 pM in 10 mM AB, and yield of 1 mg. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a upiter C4 (Phenomenex, 5pm, 300A, 150x4.6 mm) column at a flow rate of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191. l l l (SRI-230091)

[0288] FIG. 46 shows an example structure of MGS NHLF V23-4 AF647. More specifically, FIG. 46 shows the MGS_NHLF_V23-4_AF647, which included a tetramer of the MGS peptide of AFWAMILNDIYDDTPLSEFRLL conjugated to AF647. The calculated mass was 12670.71 Da. The MGS NHLF V23-4 AF647 crude peptide did not dissolve in 60% Acetonitrile.

[0289] FIG. 47 shows an example structure of MGS NHLF V28-4 AF647. More specifically, FIG. 47 shows the MGS_NHLF_V28-4_AF647, which included a tetramer of the MGS peptide of CH3CO-SFWAIANDIYD. The calculated mass was 8257.32 Da. The MGS_NHLF_V28-4_AF647 crude peptide formed a gel and was insoluble.

[0290] FIGs. 48A-48C show an example structure of MGS NHLF V29-4 AF647 and analytical results of the structure. More specifically, FIG. 48A shows the MGS NHLF V29-4 AF647, which included a tetramer of the MGS peptide of CH3CO-SFWAIANDIYDDTPLSEFRL conjugated with AF647. FIG. 48B is the resulting analytical RP-HPLC and FIG. 48C is the electrospray ionization MS results of the MGS_NHLF_V29-4_AF647. The calculated mass was 13479.07 Da, solubility of greater than 20 pM in 5 mM AB. Purity was analyzed using an Agilent 1220 Infinity HPLC system with 0.1% TFA in MQ water (eluent A) and 0.1% TFA in acetonitrile (eluent B) as mobile phases. Each injection was run through a lupiter C4 (Phenomenex, 5pm, 300A, 150X4.6 mm) column at a flow rate of ImL / minute using a 20-70% B gradient over 20 minutes at room temperature. UV absorbance at 214nm (peptide) and 651nm (AF647) were monitored.

[0291] Example embodiments in accordance with the present disclosure are directed to MGS peptides which specifically bind to lung fibroblasts and are capable of mediating internalization of cargo to lung fibroblasts. In some experiments, multimers were formed in order to mediate internalization.

Claims

PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.

191. l l l (SRI-230091)CLAIMS1. A Molecular Guidance System (MGS) peptide selected from: SEQ ID NOs: 1-29.

2. A Molecular Guidance System (MGS) peptide selected from: SEQ ID NOs: 1-2, 7-8, 10, 15, 19, 21, and 23-29.

3. The MGS peptide of claim 1 or claim 2, wherein the MGS peptide is selected from:SEQ ID NOs: 1-2, 7-8, 10, 15, 19, 21, 24, and 27-28.

4. The MGS peptide of claim 1 or claim 2, wherein the MGS peptide selectively binds to lung fibroblasts.

5. The MGS peptide of claim 1 or claim 2, wherein the MGS peptide comprises a protecting group on the N-terminus.

6. The MGS peptide of claim 5, wherein the protecting group is selected from: an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

7. The MGS peptide of claim 5, wherein the protecting group is selected from: -COCH3, -COH, -CO-(CH)n-CH3, -CO-(CH)n-NH2, y-Aminobutyric acid, pyroglutamic acid, methyl, -CO-(CH)2-CO-OH, -CO-(CH)2-CO-COH3, -CO-polyethylene glycol (PEG)X-(CH)2- NH2, -CO-PEGX-(CH)2-COH3, and CO-PEGx-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

8. The MGS peptide of claim 5, wherein the MGS peptide is selected from SEQ ID NOs: 30-58.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191.l l l (SRI-230091)9. A Molecular Guidance System (MGS) compound comprising at least two MGS peptides, wherein the at least two MGS peptides are each independently selected from SEQ ID NOs: 1-29.

10. The MGS compound of claim 9, wherein each of the at least two MGS peptides comprise a protecting group on the N-terminus selected from: an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

11. The MGS compound of claim 9, wherein the at least two MGS peptides comprise two MGS peptides.

12. The MGS compound of claim 9, wherein the at least two MGS peptides comprise four MGS peptides.

13. The MGS compound of claim 9, wherein the at least two MGS peptides are each the same or each MGS peptide of the at least two MGS peptides is different from one another.

14. The MGS compound of claim 9, wherein the MGS compound further comprises a linker structure comprising linkers selected from: a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

15. The MGS compound of claim 14, wherein the linker structure comprises: at least two PEG linkers, each respectively conjugated to one of the at least two MGS peptides, directly or indirectly; and at least one branch linker conjugated to two of the at least two PEG linkers, directly or indirectly.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.

191. l l l (SRI-230091)16. The MGS compound of claim 15, wherein the branch linker comprises a modified amino acid selected from: a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

17. The MGS compound of claim 15, further comprising a reactive group conjugated to the linker structure, directly or indirectly, and to allow for conjugation to a cargo.

18. The MGS compound of claim 17, wherein the reactive group is selected from: a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, a mal eimide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

19. The MGS compound of claim 15, wherein the linker structure further comprises additional linkers comprising reactive groups and the at least two PEG linkers are indirectly conjugated to, respectively, the one of the at least two MGS peptides through the reactive groups.

20. A Molecular Guidance System (MGS)-cargo conjugate comprising an MGS peptide conjugated to a cargo, directly or indirectly through a linker, wherein the MGS peptide is selected from SEQ ID NOs: 1-58.

21. The MGS-cargo conjugate of claim 20, wherein the MGS peptide is selected from SEQ ID NOs: 1-29, and the MGS peptide further comprises a protecting group on the N-terminus selected from: an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.

191. l l l (SRI-230091)22. The MGS-cargo conjugate of claim 20, wherein the MGS-cargo conjugate comprises a multimer that includes the MGS peptide and at least one additional MGS peptide, each of the MGS peptide and at least one additional MGS peptide being independently selected from SEQ ID NOs: 1-58.

23. A Molecular Guidance System (MGS) peptide comprising: SB1WB2B3B4NDIYD (SEQ ID NO: 59), wherein:Bi is selected from A and F;B2 is selected from A and M;B3 is selected from A and I; andB4 is selected from A and L.

24. The MGS peptide of claim 23, further comprising B5B6PB7B8B9B10B11B12 such that the MGS peptide comprises SB1WB2B3B4NDIYDB5B6PB7B8B9B10B11B12 (SEQ ID NO: 60), wherein:Bs is selected from A and D;Be is selected from A and T;B7 is selected from A and L;Bs is selected from A and S;B9 is selected from A and E;Bio is selected from A and F;Bn is selected from A and R; andB12 is selected from A and L.

25. The MGS peptide of claim 23, wherein the MGS peptide selectively binds to lung fibroblasts.

26. The MGS peptide of claim 23, wherein the MGS peptide comprises a protecting group on the N-terminus.

27. The MGS peptide of claim 26, wherein the protecting group is selected from:PCT / US24 / 55642 13 November 2024 (13.11.2024)S1647.191.l l l (SRI-230091) an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

28. The MGS peptide of claim 26, wherein the protecting group is selected from: -COCH3, -COH, -C0-(CH)n-CH3, -C0-(CH)n-NH2, y-Aminobutyric acid, pyroglutamic acid, methyl, -C0-(CH)2-C0-0H, -CO-(CH)2-CO-COH3, -CO-uoly ethylene glycol (PEG)X-(CH)2- NH2, -CO-PEGX-(CH)2-COH3, and CO-PEGx-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

29. A Molecular Guidance System (MGS) compound comprising at least two MGS peptides of any of claims 23-28.

30. A Molecular Guidance System (MGS)-cargo conjugate comprising an MGS peptide of any of claims 23-28 conjugated to a cargo, directly or indirectly through a linker.

31. A method of targeting lung fibroblasts in a subject comprising: administering the MGS compound or MGS-cargo conjugate of any one of claims9-30 to a subject.

32. The method of claim 31, wherein the MGS peptide preferentially binds to lung fibroblasts and internalizes the cargo to the lung fibroblasts.

33. The method of claim 31, wherein the cargo retains functional activity inside the lung fibroblasts.

34. The method of claim 31, wherein administering comprises an intravenous or intrathecal administration of a formulation comprising the MGS-cargo conjugate.

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